Interleukin-10 fusion proteins

ABSTRACT

The present invention generally relates to fusion proteins of antibodies and interleukin-10 (IL-10). In addition, the present invention relates to polynucleotides encoding such fusion proteins, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the fusion proteins of the invention, and to methods of using them in the treatment of disease.

FIELD OF THE INVENTION

The present invention generally relates to fusion proteins of antibodiesand interleukin-10 (IL-10). In addition, the present invention relatesto polynucleotides encoding such fusion proteins, and vectors and hostcells comprising such polynucleotides. The invention further relates tomethods for producing the fusion proteins of the invention, and tomethods of using them in the treatment of disease.

BACKGROUND

Biological Function of IL-10

IL-10 is an α-helical cytokine that is expressed as a non-covalentlylinked homodimer of ˜37 kDa. It plays a key role in the induction andmaintenance of tolerance. Its predominantly anti-inflammatory propertieshave been known for a long time. IL-10 suppresses the secretion ofpro-inflammatory cytokines like TNF α, IL-1, IL-6, IL-12 as well as Th1cytokines such as IL-2 and INFγ and controls differentiation andproliferation of macrophages, B-cells and T-cells (Glocker, E. O. etal., Ann. N.Y. Acad. Sci. 1246, 102-107 (2011); Moore, K. W. et al.,Annu. Rev. Immunol. 19, 683-765 (2001); de Waal Malefyt, R. et al., J.Exp. Med. 174, 915-924 (1991); Williams, L. M. et al., Immunology 113,281-292 (2004). Moreover, it is a potent inhibitor of antigenpresentation, inhibiting MHC II expression as well as upregulation ofco-stimulatory molecules CD80 and CD86 (Mosser, D. M. & Yhang, X.,Immunological Reviews 226, 205-218 (2008)).

Nevertheless, also immunostimulatory properties have been reported.IL-10 can costimulate B-cell activation, prolong B-cell survival, andcontribute to class switching in B-cells. Moreover, it can costimulatenatural killer (NK) cell proliferation and cytokine production and actas a growth factor to stimulate the proliferation of certain subsets ofCD8⁺ T cells (Mosser, D. M. & Yhang, X., Immunological Reviews 226,205-218 (2008); Cai, G. et al., Eur. J. Immunol. 29, 2658-2665 (1999);Santin, A. D. et al., J. Virol. 74, 4729-4737 (2000); Rowbottom, A. W.et al., Immunology 98, 80-89 (1999); Groux, H. et al., J. Immunol. 160,3188-3193 (1998)). Importantly, high doses of IL-10 (20 and 25 μg/kg,respectively) in humans can lead to an increased production of INFγ(Lauw, F. N. et al., J. Immunol. 165, 2783-2789 (2000); Tilg, H. et al.,Gut 50, 191-195 (2002)).

IL-10 signals through a two-receptor complex consisting of two copieseach of IL-10 receptor 1 (IL-10R1) and IL-10R2. IL-10R1 binds IL-10 witha relatively high affinity (˜35-200 pM) (Moore, K. W. et al., Annu. Rev.Immunol. 19, 683-765 (2001)), and the recruitment of IL-10R2 to thereceptor complex makes only a marginal contribution to ligand binding.However, the engagement of this second receptor to the complex enablessignal transduction following ligand binding. Thus, the functionalreceptor consists of a dimer of heterodimers of IL-10R1 and IL-10R2.Most hematopoietic cells constitutively express low levels of IL-10R1,and receptor expression can often be dramatically upregulated by variousstimuli. Non-hematopoietic cells, such as fibroblasts and epithelialcells, can also respond to stimuli by upregulating IL-10R1. In contrast,the IL-10R2 is expressed on most cells. The binding of IL-10 to thereceptor complex activates the Janus tyrosine kinases, JAK1 and Tyk2,associated with IL-10R1 and IL-10R2, respectively, to phosphorylate thecytoplasmic tails of the receptors. This results in the recruitment ofSTAT3 to the IL-10R1. The homodimerization of STAT3 results in itsrelease from the receptor and translocation of the phosphorylated STAThomodimer into the nucleus, where it binds to STAT3-binding elements inthe promoters of various genes. One of these genes is IL-10 itself,which is positively regulated by STAT3. STAT3 also activates thesuppressor of cytokine signaling 3 (SOCS3), which controls the qualityand quantity of STAT activation. SOCS3 is induced by IL-10 and exertsnegative regulatory effects on various cytokine genes (Mosser, D. M. &Yhang, X., Immunological Reviews 226, 205.218 (2008)).

Genetic linkage analyses and candidate gene sequencing revealed a directlink between mutations in IL-10R1 and IL-10R2 and early-onsetenterocolitis, a form of inflammatory bowel disease (IBD) (Glocker, E.O. et al., N. Engl. J. Med. 361(21), 2033-2045 (2009)). Recent datasuggest that early onset IBD can even be monogenic. Mutations in theIL-10 cytokine or its receptors lead to a loss of IL-10 function andcause severe enterocolitis in infants and small children (Glocker, E. O.et al., Ann. N.Y. Acad. Sci. 1246, 102-107 (2011)). Moreover, patientswith severe forms of Crohn's disease have a defective IL-10 productionin whole blood cell cultures and monocyte-derived dentritic cells(Correa, I. et al., J. Leukoc. Biol. 85(5), 896-903 (2009)). IBD affectsabout 1.4 million people in the United States and 2.2 million in Europe(Carter, M. J. et al., Gut 53 (Suppl. 5), V1-V16 (2004); Engel, M. A. &Neurath, M. F., J. Gastroenterol. 45, 571-583 (2010)).

Therapeutic Approaches Using IL-10

The therapeutic benefit of recombinant IL-10 in inflammatory disordersand autoimmune disease has been assessed in phase I & II clinical trialsinvestigating safety, tolerance, pharmacokinetics, pharmacodynamics,immunological and hematological effects of single or multiple dosesadministered intravenously or subcutaneously in various settings onhealthy volunteers as well as specific patient populations (Moore, K. W.et al., Annu. Rev. Immunol. 19, 683-765 (2001); Chemoff, A. E. et al.,J. Immunol. 154, 5492-5499 (1995); Huhn, R. D. et al., Blood 87, 699-705(1996); Huhn, R. D. et al., Clin. Pharmacol. Ther. 62, 171-180 (1997)).IL-10 was well tolerated without serious side effects at doses up to 25μg/kg and only mild to moderate flu-like symptoms were observed in afraction of recipients at doses up to 100 μg/kg (Moore, K. W. et al.,Annu. Rev. Immunol. 19, 683-765 (2001); Chemoff, A. E. et al., J.Immunol. 154, 5492-5499 (1995)). Tendencies towards clinical improvementwere most often seen in psoriasis (a compilation of clinical studies canbe found in Mosser, D. M. & Yhang, X., Immunological Reviews 226,205-218 (2008)), Crohn's disease (Van Deventer S. J. et al,Gastroenterology 113, 383-389 (1997); Fedorak, R. N. et al.,Gastroenterology 119, 1473-1482 (2000); Schreiber, S. et al.,Gastroenterolotgy 119, 1461-1472 (2000); Colombel J. F. et al., Gut 49,42-46 (2001)) and rheumatoid arthritis (Keystone, E. et al., Rheum. Dis.Clin. N. Am. 24, 629-639 (1998); Mosser, D. M. & Yhang, X.,Immunological Reviews 226, 205-218 (2008)).

Overall, the clinical results were unsatisfying and clinical developmentof recombinant human IL-10 which is identical to endogenous human IL-10with the exception of a methionine residue at the amino terminus(ilodecakin, TENOVIL, Schering-Plough Research Institute, Kenilworth,N.J.) was discontinued due to a lack of efficacy. A recent systematicreview of the efficacy and tolerability of recombinant human IL-10 forinduction of remission in Crohn's disease found no statisticallysignificant differences between IL-10 and placebo for complete orclinical remission and stated that patients treated with IL-10 weresignificantly more likely to withdraw from the studies due to adverseevents relative to placebo (Buruiana, F. E. et al., Cochrane DatabaseSyst. Rev. 11, CD005109 (2010)) For Crohn's disease, several reasons forthese unsatisfying results have been discussed (Herfarth, H. &Schölmerich, J., Gut 50, 146-147 (2002)): 1) local cytokineconcentrations in the gut that were too low to mediate a sustainedanti-inflammatory effect, 2) dose escalation of systemicallyadministered IL-10 was limited due to side effects, and 3) theimmunostimulatory properties of IL-10 on B cells and on INFγ productionby CD4⁺, CD8⁺, and/or natural killer cells counterbalance itsimmunosuppressive properties (Asadullah, K. et al., Pharmacol. Rev. 55,241-269 (2003); Tilg, H. et al., Gut 50, 191-195 (2002); Lauw, F. N. etal., J. Immunol. 165, 2783-2789 (2000)).

IL-10 exhibits a very short plasma half-life due to its small size of˜37 kDa which leads to rapid kidney clearance. In fact, its half life inthe systemic compartment is 2.5 h which limits the mucosalbioavailability (Braat, H. et al., Expert Opin. Biol. Ther. 3(5),725-731 (2003). In order to improve circulation time, exposure, efficacyand to reduce renal uptake, several publications report the PEGylationof this cytokine (Mattos, A. et al., J. Control Release 162, 84-91(2012); Mumm, J. B. et al., Cancer Cell 20(6), 781-796 (2011); Alvarez,H. M. et al., Drug Metab. Dispos. 40(2), 360-373 (2012)). Nevertheless,the longer systemic half-life of PEGylated non-targeted IL-10 canexacerbate known adverse events of this molecule.

It has become clear that systemic treatment using recombinant humanIL-10 is not sufficiently effective and that the focus has to be onlocal delivery of the cytokine. There are several ways to achieve thisgoal: 1) IL-10 gene therapy of immune cells, 2) genetically modified,non-pathogenic, IL-10 expression bacteria and 3) antibody-IL-10 fusionproteins in order to target the cytokine to and to accumulate thecytokine in inflamed tissues.

IL-10 gene therapy of immune cells has demonstrated effectiveness inexperimental colitis but clinical trials are hampered by concerns overthe safety of this approach for non-lethal diseases (Braat, H. et al.,Expert Opin. Biol. Ther. 3(5), 725-731 (2003)). Transgenic bacteria(Lactococcus lactis) expressing IL-10 represent an alternative route ofdelivery and the outcome of a phase I trial in Crohn's disease waspublished claiming to avoid systemic side effects due to local deliveryinto the mucosal compartment and to be biologically contained (Braat, H.et al., Gastroenterol. Hepatol. 4, 754-759 (2006); Steidler, L. et al.,Science 289, 1352-1355 (2000)). A phase IIa randomizedplacebo-controlled double-blind multi-center dose escalation study toevaluate the safety, tolerability, pharmacodynamics and efficacy ofgenetically modified Lactococcus lactis secreting human IL-10 (AG011,ActoGeniX) in patients with moderately active ulcerative colitis waswell-tolerated and safe. However, there was no significant improvementof mucosal inflammation, as measured by the modified Baron score, orclinical symptoms in patients receiving AG011 compared with placebo(Vermeire, S. et al., abstract 46 presented at the Digestive DiseaseWeek Annual Meeting in New Orleans May 2, 2010).

Antibody-cytokine fusion proteins, also called immunocytokines, offerseveral advantages in terms of drug delivery and the format of the drugitself. Local delivery of cytokines, e.g. IL-10, is achieved by fusionto antibodies or fragments thereof specific for suitable diseasemarkers. Thus, systemic side effects can be reduced and localaccumulation and retention of the compound at the site of inflammationcan be achieved. Moreover, depending on the fusion format and antibodyor antibody fragment used, properties like plasma half-life, stabilityand developability can be improved. Although an already establishedapproach in oncology, it was only recently adapted in order to treatinflammatory disorders and autoimmunity. Several cytokines (IL-10amongst others) and a photosensitizer were targeted to psoriatic lesionsby fusion to a scFv antibody fragment specific for the extra domain B offibronectin (Trachsel, E. et al., J. Invest. Dermatol. 127(4), 881-886(2007). Moreover, antibody fragments specific for the extra domain A offibronectin (F8, DEKAVIL, Philogen SpA)-IL-10 fusion proteins were usedpreclinically to inhibit the progression of established collagen-inducedarthritis (Trachsel, E. et al., Arthritis Res. Ther. 9(1), R 9 (2007);Schwager, K. et al., Arthritis Res. Ther. 11(5), R142 (2009)) andentered clinical trials. Recently, the same F8-IL-10 fusion protein wasused for targeting endometriotic lesions in a syngeneic mouse model andreduced the average lesion sizes compared to the saline control group(Schwager, K. et al., Hum. Reprod. 26(9), 2344-2352 (2011)).

The IgG-IL-10 fusion proteins of this invention have several advantagesover the known antibody fragment-based (e.g. scFv, diabody, Fab) IL-10fusion proteins, including improved produceability, stability, serumhalf-life and, surprisingly, significantly increased biological activityupon binding to target antigen.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a fusion protein of an IgG-classantibody and an IL-10 molecule, wherein the fusion protein comprises twoidentical heavy chain polypeptides and two identical light chainpolypeptides. In one embodiment, each of said heavy chain polypeptidescomprises an IgG-class antibody heavy chain and an IL-10 monomer. In amore specific embodiment, said IL-10 monomer is fused at its N-terminusto the C-terminus of said IgG-class antibody heavy chain, optionallythrough a peptide linker. In one embodiment, said heavy chainpolypeptides each essentially consist of an IgG-class antibody heavychain, an IL-10 monomer and optionally a peptide linker. In oneembodiment, each of said light chain polypeptides comprises an IgG-classantibody light chain. In one embodiment, said light chain polypeptideseach essentially consist of an IgG-class antibody light chain.

In some embodiments, said IL-10 monomer is a native IL-10 monomer,particularly a native human IL-10 monomer. In a specific embodiment,said IL-10 monomer comprises the polypeptide sequence of SEQ ID NO: 1.In one embodiment, said IL-10 monomers comprised in said heavy chainpolypeptides form a functional homodimeric IL-10 molecule.

In other embodiments, said IL-10 monomer is a modified IL-10 monomer,particularly a modified human IL-10 monomer. In one embodiment, saidmodified IL-10 monomer is stable at pH 7.0, 37° C. in monomeric form. Inone embodiment, said modified IL-10 monomer has improved stability at pH7.0, 37° C. as compared to a native IL-10 monomer. In a specificembodiment, said IL-10 monomer comprises the polypeptide sequence of SEQID NO: 5. In one embodiment, said IL-10 monomers comprised in said heavychain polypeptides do not homodimerize with each other.

In one embodiment, said IgG-class antibody comprises a modificationreducing binding affinity of the antibody to an Fc receptor, as comparedto a corresponding IgG-class antibody without said modification. In aspecific embodiment, said Fc receptor is an Fcγ receptor, particularly ahuman Fcγ receptor. In one embodiment, said Fc receptor is an activatingFc receptor, particularly an activating Fcγ receptor. In a specificembodiment, said Fc receptor is selected from the group of FcγRIIIa(CD16a), FcγRI (CD64), FcγRIIa (CD32) and Fecal (CD89). In an even morespecific embodiment, said Fc receptor is FcγIIIa, particularly humanFcγIIIa. In one embodiment, said modification reduces effector functionof the IgG-class antibody. In a specific embodiment, said effectorfunction is antibody-dependent cell-mediated cytotoxicity (ADCC). In oneembodiment, said modification is in the Fc region, particularly the CH2region, of said IgG-class antibody. In one embodiment, said IgG-classantibody comprises an amino acid substitution at position 329 (EUnumbering) of the antibody heavy chains. In a specific embodiment, saidamino acid substitution is P329G. In one embodiment, said IgG-classantibody comprises amino acid substitutions at positions 234 and 235 (EUnumbering) of the antibody heavy chains. In a specific embodiment, saidamino acid substitutions are L234A and L235A (LALA). In a particularembodiment, said IgG-class antibody comprises amino acid substitutionsL234A, L235A and P329G (EU numbering) in the antibody heavy chains.

In one embodiment, said IgG-class antibody is an IgG₁-subclass antibody.In one embodiment, said IgG-class antibody is a full-length antibody. Inone embodiment, said IgG-class antibody is a human antibody. In oneembodiment, said IgG-class antibody is a monoclonal antibody.

In one embodiment, said IgG-class antibody is capable of specificbinding to Fibroblast Activation Protein (FAP). In a specificembodiment, the fusion protein is capable of binding to FAP with anaffinity constant (K_(D)) of smaller than 1 nM, particularly smallerthan 100 pM, when measured by Surface Plasmon Resonance (SPR) at 25° C.In one embodiment, said FAP is human, mouse and/or cynomolgus FAP. In aspecific embodiment, said IgG-class antibody comprises the heavy chainCDR (HCDR) 1 of SEQ ID NO: 37, the HCDR 2 of SEQ ID NO: 41, the HCDR 3of SEQ ID NO: 49, the light chain CDR (LCDR) 1 of SEQ ID NO: 53, theLCDR 2 of SEQ ID NO: 57 and the LCDR 3 of SEQ ID NO: 61. In an even morespecific embodiment, said IgG-class antibody comprises the heavy chainvariable region (VH) of SEQ ID NO: 63 and the light chain variableregion (VL) of SEQ ID NO: 65. In another, particular, specificembodiment, said IgG-class antibody comprises the HCDR 1 of SEQ ID NO:37, the HCDR 2 of SEQ ID NO: 43, the HCDR 3 of SEQ ID NO: 47, the LCDR 1of SEQ ID NO: 51, the LCDR 2 of SEQ ID NO: 55 and the LCDR 3 of SEQ IDNO: 59. In an even more specific embodiment, said IgG-class antibodycomprises the VH of SEQ ID NO: 67 and the VL of SEQ ID NO: 69.

In one embodiment, the fusion protein is capable of binding to IL-10receptor-1 (IL-10R1) with an affinity constant (K_(D)) of smaller than 1nM, particularly smaller than 100 pM, when measured by SPR at 25° C. Ina specific embodiment, said IL-10R1 is human IL-10R1. In one embodiment,said affinity constant (K_(D)) for binding to IL-10R1 is about equal orgreater than said affinity constant (K_(D)) for binding to FAP, whenmeasured by SPR at 25° C. In a specific embodiment, said K_(D) forbinding to IL-10R1 is greater than about half of said K_(D) for bindingto FAP.

In a particular embodiment, the invention provides a fusion protein ofan IgG-class antibody and an IL-10 molecule, wherein the fusion proteincomprises two identical heavy chain polypeptides and two identical lightchain polypeptides; and wherein

(i) said IgG-class antibody comprises the heavy chain CDR (HCDR) 1 ofSEQ ID NO: 37, the HCDR 2 of SEQ ID NO: 43, the HCDR 3 of SEQ ID NO: 47,the light chain CDR (LCDR) 1 of SEQ ID NO: 51, the LCDR 2 of SEQ ID NO:55 and the LCDR 3 of SEQ ID NO: 59, or comprises the heavy chainvariable region (VH) of SEQ ID NO: 67 and the light chain variableregion (VL) of SEQ ID NO: 69;(ii) said IgG-class antibody comprises amino acid substitutions L234A,L235A and P329G (EU numbering) in the antibody heavy chains;(iii) said IL-10 molecule comprises the sequence of SEQ ID NO: 1; and(iv) said heavy chain polypeptides each comprise an IgG-class antibodyheavy chain and an IL-10 monomer fused at its N-terminus to theC-terminus of said IgG-class antibody heavy chain through a peptidelinker.

In another embodiment, the invention provides a fusion protein of anIgG-class antibody and an IL-10 molecule, wherein the fusion proteincomprises two identical heavy chain polypeptides and two identical lightchain polypeptides; and wherein

(i) said IgG-class antibody comprises the heavy chain CDR (HCDR) 1 ofSEQ ID NO: 37, the HCDR 2 of SEQ ID NO: 41, the HCDR 3 of SEQ ID NO: 49,the light chain CDR (LCDR) 1 of SEQ ID NO: 53, the LCDR 2 of SEQ ID NO:57 and the LCDR 3 of SEQ ID NO: 61, or comprises the heavy chainvariable region (VH) of SEQ ID NO: 63 and the light chain variableregion (VL) of SEQ ID NO: 65;(ii) said IgG-class antibody comprises amino acid substitutions L234A,L235A and P329G (EU numbering) in the antibody heavy chains;(iii) said IL-10 molecule comprises the sequence of SEQ ID NO: 1; and(iv) said heavy chain polypeptides each comprise an IgG-class antibodyheavy chain and an IL-10 monomer fused at its N-terminus to theC-terminus of said IgG-class antibody heavy chain through a peptidelinker.

The invention further provides a polynucleotide encoding the fusionprotein of the invention. Further provided is a vector, particularly anexpression vector, comprising the polynucleotide of the invention. Inanother aspect, the invention provides a host cell comprising thepolynucleotide or the vector of the invention. The invention alsoprovides a method for producing a fusion protein of the invention,comprising the steps of (i) culturing the host cell of the inventionunder conditions suitable for expression of the fusion protein, and (i)recovering the fusion protein. Also provided is a fusion protein of anIgG-class antibody and an IL-10 molecule produced by said method.

In one aspect, the invention provides a pharmaceutical compositioncomprising the fusion protein of the invention and a pharmaceuticallyacceptable carrier. The fusion protein or the pharmaceutical compositionof the invention is also provided for use as a medicament, and for usein the treatment or prophylaxis of an inflammatory disease, specificallyinflammatory bowel disease or rheumatoid arthritis, most specificallyinflammatory bowel disease. Further provided is the use of the fusionprotein of the invention for the manufacture of a medicament for thetreatment of a disease in an individual in need thereof, and a method oftreating a disease in an individual, comprising administering to saidindividual a therapeutically effective amount of a compositioncomprising the fusion protein of the invention in a pharmaceuticallyacceptable form. In one embodiment, said disease is an inflammatorydisease. In a more specific embodiment, said inflammatory disease isinflammatory bowel disease or rheumatoid arthritis. In an even morespecific embodiment, said inflammatory disease is inflammatory boweldisease. In one embodiment, said individual is a mammal, particularly ahuman.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of various antibody-IL-10 fusionformats. Panels (A) to (D) show formats based on an IgG antibody, panels(E) to (G) show formats based on Fab fragments. (A) “IgG-IL-10”, humanIgG (with engineered Fc-region to avoid effector functions, e.g. byamino acid substitutions L234A L235A (LALA) P329G) with one IL-10molecule (wild type human IL-10 cytokine sequence) fused to C-terminusof each IgG heavy chain (IL-10 molecules on both heavy chains dimerizewithin the same IgG molecule). Connector between heavy chain and IL-10:e.g. (G₄S)₄ 20-mer. (B) “IgG-single chain (sc) IL-10”, human IgG (withengineered Fc-region to avoid effector functions and combination of one“knob” heavy chain and one “hole” heavy chain to facilitateheterodimerization of the two) with single chain IL-10 dimer (scIL-10)fused to C-terminus of one of the IgG heavy chains. Connector betweenthe heavy chain and single chain IL-10: e.g. (G₄S)₃ 15-mer. (C)“IgG-IL-10M1”, human IgG (with engineered Fc-part to avoid effectorfunctions and combination of one “knob” heavy chain and one “hole” heavychain to facilitate heterodimerization of the two) with engineeredmonomeric IL-10 molecule fused to C-terminus of one of the IgG heavychains. Connector between the heavy chain and monomeric IL-10: e.g.(G₄S)₃ 15-mer. (D) “IgG-(IL-10M1)₂”, human IgG (with engineered Fc-partto avoid effector functions) with one IL-10 monomer fused to theC-terminus of each IgG heavy chain (monomeric IL-10 molecules on eitherheavy chain do not dimerize). Connector between the heavy chain andIL-10: e.g. (G₄S)₃ 15-mer linker. (E) “Fab-IL-10”, Fab fragment with oneIL-10 molecule (wild type human IL-10 cytokine sequence) fused toC-terminus of the Fab heavy chain (two of these fusions form ahomodimeric active molecule by dimerization via IL-10 portion).Connector between the heavy chain and IL-10: e.g. (G₄S)₃ 15-mer. (F)“Fab-scIL-10-Fab”, tandem Fab fragments intermitted by a single chainIL-10 dimer (i.e. two IL-10 molecules have been linked by e.g. a (G₄S)₄20-mer linker and inserted between the C-terminus of the first Fab heavychain (HC1) and the N-terminus of the second Fab heavy chain (HC2),resulting in a single peptide chain comprising HC1-IL-10-IL-10-HC2). Twolight chains (which can be identical to the ones used for the otherconstructs) pair with these two heavy chains. (G) “Fab-IL-10M1-Fab”,tandem Fab fragments intermitted by an engineered monomeric IL-10molecule. Apart from the monomeric IL-10 portion, this format isidentical to (F).

FIG. 2. Purification of FAP-targeted 4B9-based IgG-IL-10 construct (seeSEQ ID NOs 25 and 27). (A) Elution profile of the protein A purificationstep. (B) Elution profile of the size exclusion chromatography step. (C)Analytical SDS-PAGE (reduced (R): NuPAGE Novex Bis-Tris Mini Gel,Invitrogen, MOPS running buffer, non-reduced (NR): NuPAGE Tris-Acetate,Invitrogen, Tris-Acetate running buffer) of the final product. M: sizemarker (D) Analytical size exclusion chromatography on a Superdex 200column of the final product. Monomer content 99.8%.

FIG. 3. Purification of FAP-targeted 4G8-based IgG-scIL-10 construct(see SEQ ID NOs 7, 11 and 13). (A) Elution profile of the protein Apurification step. (B) Elution profile of the size exclusionchromatography step (desired product indicated by dotted square). (C)Analytical SDS-PAGE (reduced (R): NuPAGE Novex Bis-Tris Mini Gel,Invitrogen, MOPS running buffer, non-reduced (NR): NuPAGE Tris-Acetate,Invitrogen, Tris-Acetate running buffer) of the final product;additional lower MW-band on non-reduced gel may represent ahalf-molecule consisting of one heavy chain and light chain. (D)Analytical size exclusion chromatography on a TSKgel G3000 SW XL columnof the final product. Monomer content 80.6%.

FIG. 4. Purification of FAP-targeted 4G8-based IgG-IL-10M1 construct(see SEQ ID NOs 7, 13 and 15). (A) Elution profile of the protein Apurification step. (B) Elution profile of the size exclusionchromatography step. (C) Analytical SDS-PAGE (reduced (R): NuPAGE NovexBis-Tris Mini Gel, Invitrogen, MOPS running buffer, non-reduced (NR):NuPAGE Tris-Acetate, Invitrogen, Tris-Acetate running buffer) of thefinal product. (D) Analytical size exclusion chromatography on aSuperdex 200 column of the final product. Monomer content 98.2%.

FIG. 5. Purification of FAP-targeted 4B9-based IgG-(IL-10M1)₂ construct(see SEQ ID NOs 25 and 29). (A) Elution profile of the protein Apurification step. (B) Elution profile of the size exclusionchromatography step. (C) LabChip GX (Caliper) analysis of the finalproduct. (D) Analytical size exclusion chromatography on a TKSgel G3000SW XL column of the final product. Monomer content 100%.

FIG. 6. Purification of FAP-targeted 4B9-based Fab-IL-10 construct (seeSEQ ID NOs 25 and 31). (A) Elution profile of the protein A purificationstep. (B) Elution profile of the size exclusion chromatography step. (C)Analytical SDS-PAGE (reduced (R): NuPAGE Novex Bis-Tris Mini Gel,Invitrogen, MOPS running buffer, non-reduced (NR): NuPAGE Tris-Acetate,Invitrogen, Tris-Acetate running buffer) of the final product. (D)Analytical size exclusion chromatography on a Superdex 200 column of thefinal product. Monomer content 92.9%.

FIG. 7. Purification of FAP-targeted 4G8-based Fab-scIL-10-Fab construct(see SEQ ID NOs 7 and 21). (A) Elution profile of the protein Apurification step. (B) Elution profile of the size exclusionchromatography step. (C) Analytical SDS-PAGE (reduced (R): NuPAGE NovexBis-Tris Mini Gel, Invitrogen, MOPS running buffer, non-reduced (NR):NuPAGE Tris-Acetate, Invitrogen, Tris-Acetate running buffer) of thefinal product. (D) Analytical size exclusion chromatography on aSuperdex 200 column of the final product. Monomer content 100%.

FIG. 8. Purification of FAP-targeted 4G8-based Fab-IL-10M1-Fab fusion(see SEQ ID NOs 7 and 23). (A) Elution profile of the protein Apurification step. (B) Elution profile of the size exclusionchromatography step. (C) Analytical SDS-PAGE (reduced (R): NuPAGE NovexBis-Tris Mini Gel, Invitrogen, MOPS running buffer, non-reduced (NR):NuPAGE Tris-Acetate, Invitrogen, Tris-Acetate running buffer) of thefinal product. (D) Analytical size exclusion chromatography on aSuperdex 200 column of the final product. Monomer content 100%.

FIG. 9. SPR assay set-up on ProteOn XPR36. (A) Covalent immobilizationof anti-penta His IgG (capture agent) on GLM chip by amine couplingfollowed by capture of FAP (ligand) and subsequent injection of anti-FAPantibody-IL-10 fusion constructs (analyte). (B) Immobilization ofbiotinylated human IL-10R1 (ligand) on neutravidin-derivatized sensorchip (NLC) followed by injection of anti-FAP antibody-IL-10 fusionconstructs (analyte).

FIG. 10. Suppression of pro-inflammatory cytokine production bymonocytes by different antibody-IL-10 fusion proteins. (A-C) 4G8Fab-IL-10 (see SEQ ID NOs 7 and 19) or 4G8 IgG-IL-10 (see SEQ ID NOs 7and 9) were immobilized on cell culture plates coated with recombinanthuman FAP before monocytes and 100 ng/ml LPS as stimulus were added for24 h. Concentrations of IL-6 (A), IL-1β (B) and TNFα (C) in supernatantwere measured subsequently (n=2). (D-F) Monocytes were incubated with0-200 nM 4G8 Fab-IL-10 or 4G8 IgG-IL-10 (in solution) and 100 ng/ml LPSas stimulus for 24 h. Concentrations of IL-6 (D), IL-1β (E) and TNFα (F)in supernatant were measured subsequently (n=2).

FIG. 11. Reproduction of the results shown in FIG. 1 using two differentblood donors. (A) 4G8 Fab-IL-10 or 4G8 IgG-IL-10 were immobilized oncell culture plates coated with recombinant human FAP before monocytesand 100 ng/ml LPS as stimulus were added for 24 h. Concentrations ofIL-6 (left), IL-10 (middle) and TNFα (right) in supernatant weremeasured subsequently (each row represents a blood donor). (B) Monocyteswere incubated with 0-200 nM 4G8 Fab-IL-10, 4G8 IgG-IL-10 or wild-typehuman IL-10 (in solution) and 100 ng/ml LPS as stimulus for 24 h.Concentrations of IL-6 (left), IL-1β (middle) and TNFα (right) insupernatant were measured subsequently (each row represents a blooddonor).

FIG. 12. Suppression of IL-6 production by monocytes by differentantibody-IL-10 fusion proteins. (A-C) 4G8 Fab-IL-10, 4G8 IgG-IL-10 orcorresponding untargeted constructs were immobilized on cell cultureplates coated with recombinant human FAP before monocytes and 100 ng/mlLPS as stimulus were added for 24 h. Concentrations of IL-6 insupernatant were measured subsequently. (D-F) Monocytes were incubatedwith 0-200 nM 4G8 Fab-IL-10, 4G8 IgG-IL-10, corresponding untargetedconstructs or wild-type human IL-10 (in solution) and 100 ng/ml LPS asstimulus for 24 h. Concentrations of IL-6 in supernatant were measuredsubsequently.

FIG. 13. Suppression of IL-6 production by monocytes by differentantibody-IL-10 fusion proteins. (A-D) 4G8 Fab-IL-10 (lower row) or 4G8IgG-IL-10 (upper row) were immobilized on cell culture plates coatedwith different concentrations of recombinant human FAP before monocytesand 100 ng/ml LPS as stimulus were added for 24 h. Concentrations ofIL-6 in supernatant were measured subsequently.

FIG. 14. Suppression of IL-6 production by monocytes by differentantibody-IL-10 fusion proteins. 4G8 Fab-IL-10 (B) or 4G8 IgG-IL-10 (A)were immobilized on cell culture plates coated with differentconcentrations of recombinant human FAP before monocytes and 100 ng/mlLPS as stimulus were added for 24 h. Concentrations of IL-6 insupernatant were measured subsequently. The same data as in FIG. 13 isplotted, but in different comparison.

FIG. 15. Suppression of IL-6 production by monocytes by differentantibody-IL-10 fusion proteins. (A) 4B9 Fab-IL-10 (see SEQ ID NOs 25 and31) or 4B9 IgG-IL-10 (see SEQ ID NOs 25 and 27) constructs wereimmobilized on cell culture plates coated with recombinant human FAPbefore monocytes and 100 ng/ml LPS as stimulus were added for 24 h.Concentrations of IL-6 in supernatant were measured subsequently. (B)Monocytes were incubated with 0-200 nM 4B9 Fab-IL-10 or 4B9 IgG-IL-10constructs (in solution) and 100 ng/ml LPS as stimulus for 24 h.Concentrations of IL-6 in supernatant were measured subsequently.

FIG. 16. Comparison of size exclusion chromatography (SEC) profiles ofFab-IL-10 and IgG-IL-10 formats. Arrows indicate the desired dimericproducts, aggregates are indicated by dotted circles and monomers areindicated by solid circles. In contrast to the Fab-IL-10 format, theIgG-IL-10 format does not lead to monomers or ‘half-molecules’ due tothe disulfide-linked covalent homodimerization of its heavy chains.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Terms are used herein as generally used in the art, unless otherwisedefined in the following. “Fibroblast Activation Protein”, abbreviatedas FAP, also known as Seprase (EC 3.4.21), refers to any native FAP fromany vertebrate source, including mammals such as primates (e.g. humans),non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice andrats), unless otherwise indicated. The term encompasses “full-length,”unprocessed FAP as well as any form of FAP that results from processingin the cell. The term also encompasses naturally occurring variants ofFAP, e.g., splice variants or allelic variants. In one embodiment, theantibody of the invention is capable of specific binding to human, mouseand/or cynomolgus FAP. The amino acid sequence of human FAP is shown inUniProt (www.uniprot.org) accession no. Q12884 (version 128), or NCBI(www.ncbi.nlm.nih.gov/) RefSeq NP_004451.2. The extracellular domain(ECD) of human FAP extends from amino acid position 26 to 760. The aminoacid and nucleotide sequences of a His-tagged human FAP ECD is shown inSEQ ID NOs 81 and 82, respectively. The amino acid sequence of mouse FAPis shown in UniProt accession no. P97321 (version 107), or NCBI RefSeqNP_032012.1. The extracellular domain (ECD) of mouse FAP extends fromamino acid position 26 to 761. SEQ ID NOs 83 and 84 show the amino acidand nucleotide sequences, respectively, of a His-tagged mouse FAP ECD.SEQ ID NOs 85 and 86 show the amino acid and nucleotide sequences,respectively, of a His-tagged cynomolgus FAP ECD.

By “human IL-10R1” is meant the protein described in UniProt accessionno. Q13651 (version 115), particularly the extracellular domain of saidprotein which extends from amino acid position 22 to amino acid position235 of the full sequence. SEQ ID NOs 87 and 88 show the amino acid andnucleotide sequences, respectively, of a human IL-10R1 ECD fused to ahuman Fc region. As used herein, the term “fusion protein” refers to afusion polypeptide molecule comprising an antibody and an IL-10molecule, wherein the components of the fusion protein are linked toeach other by peptide-bonds, either directly or through peptide linkers.For clarity, the individual peptide chains of the antibody component ofthe fusion protein may be linked non-covalently, e.g. by disulfidebonds.

“Fused” refers to components that are linked by peptide bonds, eitherdirectly or via one or more peptide linkers.

By “specific binding” is meant that the binding is selective for theantigen and can be discriminated from unwanted or non-specificinteractions. The ability of an antibody to bind to a specific antigencan be measured either through an enzyme-linked immunosorbent assay(ELISA) or other techniques familiar to one of skill in the art, e.g.Surface Plasmon Resonance (SPR) technique (analyzed on a BIAcoreinstrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), andtraditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). Inone embodiment, the extent of binding of an antibody to an unrelatedprotein is less than about 10% of the binding of the antibody to theantigen as measured, e.g. by SPR. In certain embodiments, an antibodythat binds to the antigen has a dissociation constant (K_(D)) of ≦1 μM,≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g. 10⁻⁸M orless, e.g. from 10⁻⁸M to 10⁻¹³M, e.g. from 10⁻⁹M to 10⁻¹³ M).

“Affinity” or “binding affinity” refers to the strength of the sum totalof non-covalent interactions between a single binding site of a molecule(e.g. an antibody) and its binding partner (e.g. an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g. antibody and antigen). The affinity of amolecule X for its partner Y can generally be represented by thedissociation constant (K_(D)), which is the ratio of dissociation andassociation rate constants (k_(off) and k_(on), respectively). Thus,equivalent affinities may comprise different rate constants, as long asthe ratio of the rate constants remains the same. Affinity can bemeasured by common methods known in the art, including those describedherein. A particular method for measuring affinity is Surface PlasmonResonance (SPR).

“Reduced binding”, for example reduced binding to an Fc receptor, refersto a decrease in affinity for the respective interaction, as measuredfor example by SPR. For clarity the term includes also reduction of theaffinity to zero (or below the detection limit of the analytic method),i.e. complete abolishment of the interaction. Conversely, “increasedbinding” refers to an increase in binding affinity for the respectiveinteraction.

As used herein, the term “single-chain” refers to a molecule comprisingamino acid monomers linearly linked by peptide bonds.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂, diabodies, linear antibodies, single-chain antibody molecules(e.g. scFv), and single-domain antibodies. For a review of certainantibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For areview of scFv fragments, see e.g. Plückthun, in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; andU.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab andF(ab′)₂ fragments comprising salvage receptor binding epitope residuesand having increased in vivo half-life, see U.S. Pat. No. 5,869,046.Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., ProcNatl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies arealso described in Hudson et al., Nat Med 9, 129-134 (2003).Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see e.g. U.S. Pat. No. 6,248,516 B1). Antibodyfragments can be made by various techniques, including but not limitedto proteolytic digestion of an intact antibody as well as production byrecombinant host cells (e.g. E. coli or phage), as described herein.

The terms “full length antibody”, “intact antibody”, and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG-classantibodies are heterotetrameric glycoproteins of about 150,000 daltons,composed of two light chains and two heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3),also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable region (VL), also called avariable light domain or a light chain variable domain, followed by alight chain constant domain (CL), also called a light chain constantregion. The heavy chain of an antibody may be assigned to one of fivetypes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subtypes, e.g. γ₁ (IgG₁), γ₂ (IgG₂),γ₃ (IgG₃), γ₄ (IgG₄), α₁ (IgA₁) and α₂ (IgA₂). The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain. Asused herein, “Fab fragment” refers to an antibody fragment comprising alight chain fragment comprising a VL domain and a constant domain of alight chain (CL), and a VH domain and a first constant domain (CH1) of aheavy chain.

The “class” of an antibody or immunoglobulin refers to the type ofconstant domain or constant region possessed by its heavy chain. Thereare five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constantdomains that correspond to the different classes of immunoglobulins arecalled α, δ, ε, γ, and μ, respectively.

An “IgG-class antibody” refers to an antibody having the structure of anaturally occurring immunoglobulin G (IgG) molecule. The antibody heavychain of an IgG-class antibody has the domain structure VH—CH1-CH2-CH3.The antibody light chain of an IgG-class antibody has the domainstructure VL-CL. An IgG-class antibody essentially consists of two Fabfragments and an Fc domain, linked via the immunoglobulin hinge region.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). See, e.g. Kindt etal., Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91 (2007).A single VH or VL domain may be sufficient to confer antigen-bindingspecificity.

The term “hypervariable region” or “HVR”, as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe complementarity determining regions (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothiaand Lesk, J. Mol. Biol. 196, 901-917 (1987)). Exemplary CDRs (CDR-L1,CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and95-102 of H3 (Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). With the exception of CDR1 in VH, CDRs generallycomprise the amino acid residues that form the hypervariable loops. CDRsalso comprise “specificity determining residues,” or “SDRs,” which areresidues that contact antigen. SDRs are contained within regions of theCDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1,a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at aminoacid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58of H2, and 95-102 of H3 (see Almagro and Fransson, Front. Biosci. 13,1619-1633 (2008)). Unless otherwise indicated, HVR residues and otherresidues in the variable domain (e.g. FR residues) are numbered hereinaccording to Kabat et al., supra (referred to as “Kabat numbering”).

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1 (L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g. containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an antibody heavy chain that contains at least aportion of the constant region. The term includes native sequence Fcregions and variant Fc regions. An IgG Fc region comprises an IgG CH2and an IgG CH3 domain. The “CH2 domain” of a human IgG Fc region usuallyextends from an amino acid residue at about position 231 to an aminoacid residue at about position 340. In one embodiment, a carbohydratechain is attached to the CH2 domain. The CH2 domain herein may be anative sequence CH2 domain or variant CH2 domain. The “CH3 domain”comprises the stretch of residues C-terminal to a CH2 domain in an Fcregion (i.e. from an amino acid residue at about position 341 to anamino acid residue at about position 447 of an IgG). The CH3 regionherein may be a native sequence CH3 domain or a variant CH3 domain (e.g.a CH3 domain with an introduced “protuberance” (“knob”) in one chainthereof and a corresponding introduced “cavity” (“hole”) in the otherchain thereof; see U.S. Pat. No. 5,821,333, expressly incorporatedherein by reference). Such variant CH3 domains may be used to promoteheterodimerization of two non-identical antibody heavy chains as hereindescribed. In one embodiment, a human IgG heavy chain Fc region extendsfrom Cys226, or from Pro230, to the carboxyl-terminus of the heavychain. However, the C-terminal lysine (Lys447) of the Fc region may ormay not be present. Unless otherwise specified herein, numbering ofamino acid residues in the Fc region or constant region is according tothe EU numbering system, also called the EU index, as described in Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., 1991.

The term “effector functions” refers to those biological activitiesattributable to the Fc region of an antibody, which vary with theantibody isotype. Examples of antibody effector functions include: C1qbinding and complement dependent cytotoxicity (CDC), Fc receptorbinding, antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), cytokine secretion,immune complex-mediated antigen uptake by antigen presenting cells, downregulation of cell surface receptors (e.g. B cell receptor), and B cellactivation.

An “activating Fc receptor” is an Fc receptor that following engagementby an Fc region of an antibody elicits signaling events that stimulatethe receptor-bearing cell to perform effector functions. Activating Fcreceptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), andFcαRI (CD89). A particular activating Fc receptor is human FcγRIIIa (seeUniProt accession no. P08637 (version 141)).

By a “native IL-10”, also termed “wild-type IL-10”, is meant a naturallyoccurring IL-10, as opposed to a “modified IL-10”, which has beenmodified from a naturally occurring IL-10, e.g. to alter one or more ofits properties such as stability. A modified IL-10 molecule may forexample comprise modifications in the amino acid sequence, e.g. aminoacid substitutions, deletions or insertions. A particular modified IL-10molecule with increased stability in monomeric form has been describedby Josephson et al. (J Biol Chem 275, 13552-13557 (2000)).

Native IL-10 is a homodimer composed of two α-helical, monomericdomains. The sequence of a native human IL-10 monomeric domain is shownin SEQ ID NO: 1. Hence, an “IL-10 monomer” is a protein of substantiallysimilar sequence and/or structure as a monomeric domain of native IL-10.

By “stable” or “stability” when used with reference to a protein ismeant that the structural integrity of the protein (e.g. its secondarystructure) is preserved.

By “functional” when used with reference to a protein is meant that theprotein is able to mediate biological functions, particularly thebiological functions that a corresponding protein occurring in nature(e.g. native IL-10) would mediate. In the case of IL-10, biologicalfunctions may include activation of IL-10 receptor signaling,suppression of secretion of pro-inflammatory cytokines such as TNF α,IL-1, IL-6, IL-12, IL-2 and/or INFγ, inhibition of MHC II expression andupregulation of co-stimulatory molecules such as CD80 and/or CD86 incells expressing IL-10 receptors (e.g. monocytes).

The term “peptide linker” refers to a peptide comprising one or moreamino acids, typically about 2-20 amino acids. Peptide linkers are knownin the art or are described herein. Suitable, non-immunogenic linkerpeptides include, for example, (G₄S)_(n), (SG₄)_(n) or G₄(SG₄)_(n)peptide linkers. “n” is generally a number between 1 and 10, typicallybetween 2 and 4.

A “knob-into-hole modification” refers to a modification within theinterface between two antibody heavy chains in the CH3 domain, whereini) in the CH3 domain of one heavy chain, an amino acid residue isreplaced with an amino acid residue having a larger side chain volume,thereby generating a protuberance (“knob”) within the interface in theCH3 domain of one heavy chain which is positionable in a cavity (“hole”)within the interface in the CH3 domain of the other heavy chain, and ii)in the CH3 domain of the other heavy chain, an amino acid residue isreplaced with an amino acid residue having a smaller side chain volume,thereby generating a cavity (“hole”) within the interface in the secondCH3 domain within which a protuberance (“knob”) within the interface inthe first CH3 domain is positionable. In one embodiment, the“knob-into-hole modification” comprises the amino acid substitutionT366W and optionally the amino acid substitution S354C in one of theantibody heavy chains, and the amino acid substitutions T366S, L368A,Y407V and optionally Y349C in the other one of the antibody heavychains. The knob-into-hole technology is described e.g. in U.S. Pat. No.5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621(1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, themethod involves introducing a protuberance (“knob”) at the interface ofa first polypeptide and a corresponding cavity (“hole”) in the interfaceof a second polypeptide, such that the protuberance can be positioned inthe cavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine). Introduction of two cysteineresidues at position 5354 and Y349, respectively, results in formationof a disulfide bridge between the two antibody heavy chains in the Fcregion, further stabilizing the dimer (Carter, J Immunol Methods 248,7-15 (2001)).

An amino acid “substitution” refers to the replacement in a polypeptideof one amino acid with another amino acid. In one embodiment, an aminoacid is replaced with another amino acid having similar structuraland/or chemical properties, e.g., conservative amino acid replacements.“Conservative” amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Non-conservative substitutions will entail exchanging amember of one of these classes for another class. For example, aminoacid substitutions can also result in replacing one amino acid withanother amino acid having different structural and/or chemicalproperties, for example, replacing an amino acid from one group (e.g.,polar) with another amino acid from a different group (e.g., basic).Amino acid substitutions can be generated using genetic or chemicalmethods well known in the art. Genetic methods may include site-directedmutagenesis, PCR, gene synthesis and the like. It is contemplated thatmethods of altering the side chain group of an amino acid by methodsother than genetic engineering, such as chemical modification, may alsobe useful. Various designations may be used herein to indicate the sameamino acid substitution. For example, a substitution from proline atposition 329 of the antibody heavy chain to glycine can be indicated as329G, G329, G₃₂₉, P329G, or Pro329Gly.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary. In situations where ALIGN-2 is employed for amino acidsequence comparisons, the % amino acid sequence identity of a givenamino acid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

“Polynucleotide” or “nucleic acid” as used interchangeably herein,refers to polymers of nucleotides of any length, and include DNA andRNA. The nucleotides can be deoxyribonucleotides, ribonucleotides,modified nucleotides or bases, and/or their analogs, or any substratethat can be incorporated into a polymer by DNA or RNA polymerase or by asynthetic reaction. A polynucleotide may comprise modified nucleotides,such as methylated nucleotides and their analogs. A sequence ofnucleotides may be interrupted by non-nucleotide components. Apolynucleotide may comprise modification(s) made after synthesis, suchas conjugation to a label.

The term “modification” refers to any manipulation of the peptidebackbone (e.g. amino acid sequence) or the post-translationalmodifications (e.g. glycosylation) of a polypeptide.

The term “vector” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors”.

The terms “host cell”, “host cell line”, and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.A host cell is any type of cellular system that can be used to generatethe fusion proteins of the present invention. Host cells includecultured cells, e.g. mammalian cultured cells, such as CHO cells, BHKcells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myelomacells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insectcells, and plant cells, to name only a few, but also cells comprisedwithin a transgenic animal, transgenic plant or cultured plant or animaltissue.

An “effective amount” of an agent refers to the amount that is necessaryto result in a physiological change in the cell or tissue to which it isadministered.

A “therapeutically effective amount” of an agent, e.g. a pharmaceuticalcomposition, refers to an amount effective, at dosages and for periodsof time necessary, to achieve the desired therapeutic or prophylacticresult. A therapeutically effective amount of an agent for exampleeliminates, decreases, delays, minimizes or prevents adverse effects ofa disease.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g. humans and non-human primates such as monkeys),rabbits, and rodents (e.g. mice and rats). Particularly, the individualor subject is a human.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical composition, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of a disease in the individual being treated,and can be performed either for prophylaxis or during the course ofclinical pathology. Desirable effects of treatment include, but are notlimited to, preventing occurrence or recurrence of disease, alleviationof symptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

Fusion Proteins of the Invention

The invention provides novel antibody-IL-10 fusion protein withparticularly advantageous properties such as produceability, stability,binding affinity and biological activity.

In a first aspect, the invention provides a fusion protein of anIgG-class antibody and an IL-10 molecule, wherein the fusion proteincomprises two identical heavy chain polypeptides and two identical lightchain polypeptides. In one embodiment, each of said heavy chainpolypeptides comprises an IgG-class antibody heavy chain and an IL-10monomer. In a more specific embodiment, said IL-10 monomer is fused atits N-terminus to the C-terminus of said IgG-class antibody heavy chain,optionally through a peptide linker. In one embodiment, said heavy chainpolypeptides each essentially consist of an IgG-class antibody heavychain, an IL-10 monomer and optionally a peptide linker. In oneembodiment, each of said light chain polypeptides comprises an IgG-classantibody light chain. In one embodiment, said light chain polypeptideseach essentially consist of an IgG-class antibody light chain. Ascompared to fusion proteins based on antibody fragments, the presence ofan IgG-class antibody confers to the fusion protein of the inventionfavorable pharmacokinetic properties including a prolonged serumhalf-life (due to recycling through binding to FcRn, and molecular sizebeing well above the threshold for renal filtration). The presence of anIgG-class antibody also enables simple purification of fusion proteinsby e.g. protein A affinity chromatography. Surprisingly, as shown in theexamples comparing the IgG-based IgG-IL-10 fusion protein of theinvention to a corresponding fusion protein based on Fab fragments(Fab-IL-10), the presence of an IgG-class antibody also improvesbiological activity of the fusion protein when bound to its targetantigen. The use of identical heavy (and light) chain polypeptidesallows for simple production of the fusion protein, avoiding theformation of undesired side products and obviating the need formodifications promoting heterodimerization of non-identical heavychains, such as a knob-into-hole modification.

In some embodiments, said IL-10 monomer is a native IL-10 monomer,particularly a native human IL-10 monomer. In a specific embodiment,said IL-10 monomer comprises the polypeptide sequence of SEQ ID NO: 1.In one embodiment, said IL-10 monomers comprised in said heavy chainpolypeptides form a functional homodimeric IL-10 molecule. This fusionprotein format is particularly advantageous in that the two IL-10monomers form a fully functional, biologically active IL-10 dimer.Moreover, in contrast to fusion proteins based on antibody fragments, inthe fusion protein of the invention dimerization not only occurs inbetween the IL-10 monomers, but also between the antibody heavy chainsto which the monomers are fused. Therefore, the tendency of the IL-10dimer comprised the fusion proteins of the invention of disassemblinginto two monomers is reduced, as compared e.g. to the Fab-IL-10 fusionproteins described herein (see FIG. 16). Importantly, this fusionprotein format is also superior to other fusion protein formatsdescribed herein in terms of biological activity.

In other embodiments, said IL-10 monomer is a modified IL-10 monomer,particularly a modified human IL-10 monomer. In one embodiment, saidmodified IL-10 monomer is stable at pH 7.0, 37° C. in monomeric form. Inone embodiment, said modified IL-10 monomer has improved stability at pH7.0, 37° C. as compared to a native IL-10 monomer. A suitable modifiedIL-10 monomer is described in Josephson et al., J Biol Chem 275,13552-13557 (2000), which also describes methods for measuring stabilityof IL-10 monomers. In a specific embodiment, said IL-10 monomercomprises the polypeptide sequence of SEQ ID NO: 5. In one embodiment,said IL-10 monomers comprised in said heavy chain polypeptides do nothomodimerize with each other. This fusion protein format comprises twoseparate modified IL-10 monomers, rather than an IL-10 dimer. As shownin the examples, this fusion protein format has similar binding affinity(avidity) to the IL-10 receptor 1 as the fusion proteins comprising anIL-10 dimer. Moreover, it is particularly well produceable, with highexpression yields and little aggregation propensity.

In one embodiment, said IgG-class antibody is an IgG₁-subclass antibody.In one embodiment, said IgG-class antibody is a human antibody, i.e. itcomprises human variable and constant regions. Sequences of exemplaryhuman IgG₁ heavy and light chain constant regions are shown in SEQ IDNOs 79 and 80, respectively. In one embodiment, the IgG-class antibodycomprises a human Fc region, particularly a human IgG Fc region, moreparticularly a human IgG₁ Fc region. In one embodiment, said IgG-classantibody is a full-length antibody. In one embodiment, said IgG-classantibody is a monoclonal antibody.

While the Fc domain of the IgG-class antibody confers to the fusionproteins favorable pharmacokinetic properties, including a long serumhalf-life which contributes to good accumulation in the target tissueand a favorable tissue-blood distribution ratio, it may at the same timelead to undesirable targeting of the fusion protein to cells expressingFc receptors rather than to the preferred antigen-bearing cells.Moreover, the activation of Fc receptor signaling pathways may lead tocytokine release resulting in activation of (pro-inflammatory) cytokinereceptors and severe side effects upon systemic administration.Therefore, in one embodiment, said IgG-class antibody comprises amodification reducing binding affinity of the antibody to an Fcreceptor, as compared to a corresponding IgG-class antibody without saidmodification. In a specific embodiment, said Fc receptor is an Fcγreceptor, particularly a human Fcγ receptor. Binding affinity to Fcreceptors can be easily determined e.g. by ELISA, or by Surface PlasmonResonance (SPR) using standard instrumentation such as a BIAcoreinstrument (GE Healthcare) and Fc receptors such as may be obtained byrecombinant expression. A specific illustrative and exemplary embodimentfor measuring binding affinity is described in the following. Accordingto one embodiment, Binding affinity to an Fc receptor is measured bysurface plasmon resonance using a BIACORE® T100 machine (GE Healthcare)at 25° C. with ligand (Fc receptor) immobilized on CM5 chips. Briefly,carboxymethylated dextran biosensor chips (CM5, GE Healthcare) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Recombinant ligand is diluted with 10 mM sodiumacetate, pH 5.5, to 0.5-30 μg/ml before injection at a flow rate of 10μl/min to achieve approximately 100-5000 response units (RU) of coupledprotein. Following the injection of the ligand, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, three- tofive-fold serial dilutions of antibody (range between ˜0.01 nM to 300nM) are injected in HBS-EP+ (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3mM EDTA, 0.05% Surfactant P20, pH 7.4) at 25° C. at a flow rate ofapproximately 30-50 μl/min. Association rates (k_(on)) and dissociationrates (k_(off)) are calculated using a simple one-to-one Langmuirbinding model (BIACORE® T100 Evaluation Software version 1.1.1) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (K_(D)) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J Mol Biol 293, 865-881 (1999).Alternatively, binding affinity antibodies to Fc receptors may beevaluated using cell lines known to express particular Fc receptors,such as NK cells expressing Fcγ IIIa receptor.

In one embodiment, the modification comprises one or more amino acidmutation that reduces the binding affinity of the antibody to an Fcreceptor. In one embodiment the amino acid mutation is an amino acidsubstitution. Typically, the same one or more amino acid mutation ispresent in each of the two antibody heavy chains. In one embodiment saidamino acid mutation reduces the binding affinity of the antibody to theFc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. Inembodiments where there is more than one amino acid mutation thatreduces the binding affinity of the antibody to the Fc receptor, thecombination of these amino acid mutations may reduce the bindingaffinity of the antibody to the Fc receptor by at least 10-fold, atleast 20-fold, or even at least 50-fold. In one embodiment saidIgG-class antibody exhibits less than 20%, particularly less than 10%,more particularly less than 5% of the binding affinity to an Fc receptoras compared to a corresponding IgG-class antibody without saidmodification.

In one embodiment, said Fc receptor is an activating Fc receptor. In aspecific embodiment, said Fc receptor is selected from the group ofFcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32) and FcαRI (CD89). In aspecific embodiment the Fc receptor is an Fcγ receptor, morespecifically an FcγRIIIa, FcγRI or FcγRIIa receptor. Preferably, bindingaffinity to each of these receptors is reduced. In an even more specificembodiment, said Fc receptor is FcγIIIa, particularly human FcγIIIa. Insome embodiments binding affinity to a complement component,specifically binding affinity to C1q, is also reduced. In one embodimentbinding affinity to neonatal Fc receptor (FcRn) is not reduced.Substantially similar binding to FcRn, i.e. preservation of the bindingaffinity of the antibody to said receptor, is achieved when the antibodyexhibits greater than about 70% of the binding affinity of an unmodifiedform of the antibody to FcRn. IgG-class antibodies comprised in thefusion proteins of the invention may exhibit greater than about 80% andeven greater than about 90% of such affinity.

In one embodiment, said modification reducing binding affinity of theantibody to an Fc receptor is in the Fc region, particularly the CH2region, of the IgG-class antibody. In one embodiment, said IgG-classantibody comprises an amino acid substitution at position 329 (EUnumbering) of the antibody heavy chains. In a more specific embodimentsaid amino acid substitution is P329A or P329G, particularly P329G. Inone embodiment, said IgG-class antibody comprises amino acidsubstitutions at positions 234 and 235 (EU numbering) of the antibodyheavy chains. In a specific embodiment, said amino acid substitutionsare L234A and L235A (LALA). In one embodiment said IgG-class antibodycomprises an amino acid substitution at position 329 (EU numbering) ofthe antibody heavy chains and a further amino acid substitution at aposition selected from position 228, 233, 234, 235, 297 and 331 of theantibody heavy chains. In a more specific embodiment the further aminoacid substitution is S228P, E233P, L234A, L235A, L235E, N297A, N297D orP331S. In a particular embodiment, said IgG-class antibody comprisesamino acid substitutions at positions P329, L234 and L235 (EU numbering)of the antibody heavy chains. In a more particular embodiment, saidIgG-class antibody comprises the amino acid substitutions L234A, L235Aand P329G (LALA P329G) in the antibody heavy chains. This combination ofamino acid substitutions almost particularly efficiently abolishes Fcγreceptor binding of a human IgG-class antibody, as described in PCTpublication no. WO 2012/130831, incorporated herein by reference in itsentirety. PCT publication no. WO 2012/130831 also describes methods ofpreparing such modified antibody and methods for determining itsproperties such as Fc receptor binding or effector functions.

Antibodies comprising modifications in the antibody heavy chains can beprepared by amino acid deletion, substitution, insertion or modificationusing genetic or chemical methods well known in the art. Genetic methodsmay include site-specific mutagenesis of the encoding DNA sequence, PCR,gene synthesis, and the like. The correct nucleotide changes can beverified for example by sequencing.

Antibodies which comprise modifications reducing Fc receptor bindinggenerally have reduced effector functions, particularly reduced ADCC, ascompared to corresponding unmodified antibodies. Hence, in oneembodiment, said modification reducing binding affinity of the IgG-classantibody to an Fc receptor reduces effector function of the IgG-classantibody. In a specific embodiment, said effector function isantibody-dependent cell-mediated cytotoxicity (ADCC). In one embodiment,ADCC is reduced to less than 20% of the ADCC induced by a correspondingIgG-class antibody without said modification. Effector function of anantibody can be measured by methods known in the art. Examples of invitro assays to assess ADCC activity of a molecule of interest aredescribed in U.S. Pat. No. 5,500,362; Hellstrom et al. Proc Natl AcadSci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad SciUSA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337; Bruggemann et al., JExp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assaysmethods may be employed (see, for example, ACTI™ non-radioactivecytotoxicity assay for flow cytometry (CellTechnology, Inc. MountainView, Calif.); and CytoTox 96® non-radioactive cytotoxicity assay(Promega, Madison, Wis.)). Useful effector cells for such assays includeperipheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g. in a animal model such as thatdisclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).In some embodiments binding of the IgG-class antibody to a complementcomponent, specifically to C1q, is also reduced. Accordingly,complement-dependent cytotoxicity (CDC) may also be reduced. C1q bindingassays may be carried out to determine whether the antibody is able tobind C1q and hence has CDC activity. See e.g. C1q and C3c binding ELISAin WO 2006/029879 and WO 2005/100402. To assess complement activation, aCDC assay may be performed (see, for example, Gazzano-Santoro et al., JImmunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052(2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

In addition to the IgG-class antibodies described hereinabove and in PCTpublication no. WO 2012/130831, antibodies with reduced Fc receptorbinding and/or effector function also include those with substitution ofone or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329(U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants withsubstitutions at two or more of amino acid positions 265, 269, 270, 297and 327, including the so-called “DANA” Fc mutant with substitution ofresidues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

IgG₄-subclass antibodies exhibit reduced binding affinity to Fcreceptors and reduced effector functions as compared to IgG₁ antibodies.Hence, in some embodiments, said IgG-class antibody comprised in thefusion protein of the invention is an IgG₄-subclass antibody,particularly a human IgG₄-subclass antibody. In one embodiment saidIgG₄-subclass antibody comprises amino acid substitutions in the Fcregion at position 5228, specifically the amino acid substitution S228P.To further reduce its binding affinity to an Fc receptor and/or itseffector function, in one embodiment, said IgG₄-subclass antibodycomprises an amino acid substitution at position L235, specifically theamino acid substitution L235E. In another embodiment, said IgG₄-subclassantibody comprises an amino acid substitution at position P329,specifically the amino acid substitution P329G. In a particularembodiment, said IgG₄-subclass antibody comprises amino acidsubstitutions at positions S228, L235 and P329, specifically amino acidsubstitutions S228P, L235E and P329G. Such modified IgG₄-subclassantibodies and their Fcγ receptor binding properties are described inPCT publication no. WO 2012/130831, incorporated herein by reference inits entirety.

The antibodies of the invention combine a number of properties which areparticularly advantageous, for example for therapeutic applications.

In one embodiment, said IgG-class antibody is capable of specificbinding to Fibroblast Activation Protein (FAP). FAP has been identifiedas a suitable target for the treatment of inflammatory diseases usingthe fusion proteins of the invention. In a specific embodiment, thefusion protein is capable of binding to FAP with an affinity constant(K_(D)) of smaller than 1 nM, particularly smaller than 100 pM, whenmeasured by Surface Plasmon Resonance (SPR) at 25° C. A method formeasuring binding affinity to FAP by SPR is described herein. In oneembodiment, affinity (K_(D)) of fusion proteins is measured by SPR usinga ProteOn XPR36 instrument (Biorad) at 25° C. with His-tagged FAPantigens immobilized by anti-His antibodies covalently coupled to GLMchips. In an exemplary method, the target protein (PAP) is captured viaits H6-tag by a covalently immobilized anti-penta His IgG (Qiagen#34660, mouse monoclonal antibody), immobilized at high levels (up to˜5.000 RU) at 30 μl/min onto separate vertical channels of a GLM chip bysimultaneously activating all channels for 5 min with a freshly preparedmixture of 1-ethyl-3-(3-dimethylaminopropyl)-carboiimide (EDC) andN-hydroxysuccinimide (sNHS), and subsequently injecting 15 μg/mlanti-penta His IgG in 10 mM sodium acetate buffer pH 4.5 for 180 sec.Channels are blocked using a 5-min injection of ethanolamine.His6-tagged FAP is captured from a 5 μg/ml dilution in running bufferalong the vertical channels for 60 s at 30 μl/min to achieve liganddensities between ˜250 and 600 RU. In a one-shot kinetic assay set-up(OSK), fusion protein are injected as analytes along the horizontalchannels in a five-fold dilution series ranging from 50 to 0.08 nM at100 μl/min. Association phase is recorded for 180 s, dissociation phasefor 600 s. In case of interactions exhibiting very slow off-rates,recording of off-rates is extended up to 1800 s in order to observe thedissociation of the complex. Running buffer (PBST) is injected along thesixth channel to provide an “in-line” blank for referencing. Associationrates (k_(on)) and dissociation rates (k_(off)) are calculated using asimple 1:1 Langmuir binding model (ProteOn Manager software version 2.1)by simultaneously fitting the association and dissociation sensorgrams.The equilibrium dissociation constant (K_(D)) is calculated as the ratiok_(off)/k_(on).

In one embodiment, said FAP is human, mouse and/or cynomolgus FAP.Preferably, the IgG-class antibody comprised in the fusion protein ofthe invention is cross-reactive for human and cynomolgus monkey and/ormouse FAP, which enables e.g. in vivo studies in cynomolgus monkeysand/or mice prior to human use.

In a specific embodiment, said IgG-class antibody comprises the heavychain CDR (HCDR) 1 of SEQ ID NO: 37, the HCDR 2 of SEQ ID NO: 41, theHCDR 3 of SEQ ID NO: 49, the light chain CDR (LCDR) 1 of SEQ ID NO: 53,the LCDR 2 of SEQ ID NO: 57 and the LCDR 3 of SEQ ID NO: 61. In an evenmore specific embodiment, said IgG-class antibody comprises the heavychain variable region (VH) of SEQ ID NO: 63 and the light chain variableregion (VL) of SEQ ID NO: 65. In another, particular, specificembodiment, said IgG-class antibody comprises the HCDR 1 of SEQ ID NO:37, the HCDR 2 of SEQ ID NO: 43, the HCDR 3 of SEQ ID NO: 47, the LCDR 1of SEQ ID NO: 51, the LCDR 2 of SEQ ID NO: 55 and the LCDR 3 of SEQ IDNO: 59. In an even more specific embodiment, said IgG-class antibodycomprises the VH of SEQ ID NO: 67 and the VL of SEQ ID NO: 69. As shownin the examples, these antibodies show particularly strong bindingaffinity/avidity to human, mouse as well as cynomolgus FAP.

In further specific embodiments, said IgG-class antibody comprises theHCDR 1 of SEQ ID NO: 39, the HCDR 2 of SEQ ID NO: 45, the HCDR 3 of SEQID NO: 49, the light chain CDR (LCDR) 1 of SEQ ID NO: 53, the LCDR 2 ofSEQ ID NO: 57 and the LCDR 3 of SEQ ID NO: 61. In an even more specificembodiment, said IgG-class antibody comprises the VH of SEQ ID NO: 71and the VL of SEQ ID NO: 73. In another specific embodiment, saidIgG-class antibody comprises the HCDR 1 of SEQ ID NO: 37, the HCDR 2 ofSEQ ID NO: 41, the HCDR 3 of SEQ ID NO: 47, the LCDR 1 of SEQ ID NO: 51,the LCDR 2 of SEQ ID NO: 55 and the LCDR 3 of SEQ ID NO: 59. In an evenmore specific embodiment, said IgG-class antibody comprises the VH ofSEQ ID NO: 75 and the VL of SEQ ID NO: 77.

In one embodiment, the fusion protein is capable of binding to IL-10receptor-1 (IL-10R1) with an affinity constant (K_(D)) of smaller than 1nM, particularly smaller than 100 pM, when measured by SPR at 25° C. Amethod for measuring binding affinity to IL-10R1 by SPR is describedherein. In one embodiment, affinity (K_(D)) of fusion proteins ismeasured by SPR using a ProteOn XPR36 instrument (Biorad) at 25° C. withbiotinylated IL-10R1 immobilized on NLC chips by neutravidin capture. Inan exemplary method, between 400 and 1600 RU of IL-10R1 are captured onthe neutravidin-derivatized chip matrix along vertical channels at aconcentration of 10 μg/ml and a flow rate of 30 μl/sec for varyingcontact times. Binding to biotinylated IL10R1 is measured at sixdifferent analyte concentrations (50, 10, 2, 0.4, 0.08, 0 nM) byinjections in horizontal orientation at 100 μl/min, recording theassociation rate for 180 s, the dissociation rate for 600 s. Runningbuffer (PBST) is injected along the sixth channel to provide an“in-line” blank for referencing. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple 1:1 Langmuirbinding model (ProteOn Manager software version 2.1) by simultaneouslyfitting the association and dissociation sensorgrams. The equilibriumdissociation constant (K_(D)) is calculated as the ratio k_(off)/k_(on).

In a specific embodiment, said IL-10R1 is human IL-10R1. In oneembodiment, said affinity constant (K_(D)) for binding to IL-10R1 isabout equal or greater than said affinity constant (K_(D)) for bindingto FAP, when measured by SPR at 25° C. In a specific embodiment, saidK_(D) for binding to IL-10R1 is greater than about half of said K_(D)for binding to FAP. The particular ratio of KD values of the fusionprotein of the invention for binding to FAP and IL-10R1 makes themparticularly suitable for efficient targeting IL-10 to FAP-expressingtissues. Without wishing to be bound by theory, the fusion proteins ofthe invention, due to their binding affinity to FAP being at leastequally high as their binding affinity to IL-10R1, are less likely tobind to IL-10R1-expressing cells outside the target tissue (e.g. in thecirculation) prior to reaching the FAP-expressing target tissue.

In a particular aspect, the invention provides a fusion protein of ahuman IgG₁-subclass antibody, capable of specific binding to FAP andcomprising a modification reducing binding affinity of the antibody toan Fc receptor as compared to a corresponding human IgG₁-subclassantibody without said modification, and an IL-10 molecule,

wherein the fusion protein comprises two identical heavy chainpolypeptides, each comprising a native IL-10 monomer fused at itsN-terminus to the C-terminus of a human IgG₁-subclass antibody heavychain, and two identical light chains.

In a specific embodiment, said fusion protein comprises a heavy chainpolypeptide that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or 100% identical to the polypeptide of SEQ ID NO: 9, and a lightchain polypeptide that is at least about 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to the polypeptide of SEQ ID NO: 7. Inanother specific embodiment, said fusion protein comprises a heavy chainpolypeptide that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or 100% identical to the polypeptide of SEQ ID NO: 27, and a lightchain polypeptide that is at least about 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to the polypeptide of SEQ ID NO: 25.

In a further aspect, the invention provides a fusion protein of a humanIgG₁-subclass antibody, capable of specific binding to FAP andcomprising a modification reducing binding affinity of the antibody toan Fc receptor as compared to a corresponding human IgG₁-subclassantibody without said modification, and an IL-10 molecule,

wherein the fusion protein comprises two identical heavy chainpolypeptides, each comprising a modified IL-10 monomer fused at itsN-terminus to the C-terminus of a human IgG₁-subclass antibody heavychain, and two identical light chains.

In a specific embodiment, said fusion protein comprises a heavy chainpolypeptide that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or 100% identical to the polypeptide of SEQ ID NO: 17, and a lightchain polypeptide that is at least about 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to the polypeptide of SEQ ID NO: 7. Inanother specific embodiment, said fusion protein comprises a heavy chainpolypeptide that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or 100% identical to the polypeptide of SEQ ID NO: 29, and a lightchain polypeptide that is at least about 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to the polypeptide of SEQ ID NO: 25.

Polynucleotides

The invention further provides polynucleotides encoding a fusion asdescribed herein or an antigen-binding fragment thereof.

Polynucleotides of the invention include those that are at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequences set forth in SEQ ID NOs 2, 6, 8, 10, 18, 26, 28, 30, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78 and 89 including functional fragments or variants thereof.

The polynucleotides encoding fusion proteins of the invention may beexpressed as a single polynucleotide that encodes the entire fusionprotein or as multiple (e.g., two or more) polynucleotides that areco-expressed. Polypeptides encoded by polynucleotides that areco-expressed may associate through, e.g., disulfide bonds or other meansto form a functional fusion protein. For example, the light chainportion of an antibody may be encoded by a separate polynucleotide fromthe heavy chain portion of the antibody. When co-expressed, the heavychain polypeptides will associate with the light chain polypeptides toform the antibody.

In one embodiment, the present invention is directed to a polynucleotideencoding a fusion protein of an IgG-class antibody and an IL-10molecule, or an antigen-binding fragment thereof, wherein thepolynucleotide comprises a sequence that encodes a variable regionsequence as shown in SEQ ID NO 63, 65, 67, 69, 71, 73, 75 or 77. Inanother embodiment, the present invention is directed to apolynucleotide encoding a fusion protein of an IgG-class antibody and anIL-10 molecule, or a fragment thereof, wherein the polynucleotidecomprises a sequence that encodes a polypeptide sequence as shown in SEQID NO 7, 9, 17, 25, 27 or 29. In another embodiment, the invention isfurther directed to a polynucleotide encoding a fusion protein of anIgG-class antibody and an IL-10 molecule, or a fragment thereof, whereinthe polynucleotide comprises a sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequenceshown SEQ ID NO 2, 6, 8, 10, 18, 26, 28, 30, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78 or 89. In anotherembodiment, the invention is directed to a polynucleotide encoding afusion protein of an IgG-class antibody and an IL-10 molecule, or afragment thereof, wherein the polynucleotide comprises a nucleic acidsequence shown in SEQ ID NO 2, 6, 8, 10, 18, 26, 28, 30, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78 or89. In another embodiment, the invention is directed to a polynucleotideencoding a fusion protein of an IgG-class antibody and an IL-10molecule, or a fragment thereof, wherein the polynucleotide comprises asequence that encodes a variable region sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acidsequence of SEQ ID NO 63, 65, 67, 69, 71, 73, 75 or 77. In anotherembodiment, the invention is directed to a polynucleotide encoding afusion protein of an IgG-class antibody and an IL-10 molecule, or afragment thereof, wherein the polynucleotide comprises a sequence thatencodes a polypeptide sequence that is at least 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO 7, 9,17, 25, 27 or 29. The invention encompasses a polynucleotide encoding ana fusion protein of an IgG-class antibody and an IL-10 molecule, or afragment thereof, wherein the polynucleotide comprises a sequence thatencodes the variable region sequences of SEQ ID NO 63, 65, 67, 69, 71,73, 75 or 77 with conservative amino acid substitutions. The inventionalso encompasses a polynucleotide encoding a fusion protein of anIgG-class antibody and an IL-10 molecule, or a fragment thereof, whereinthe polynucleotide comprises a sequence that encodes the polypeptidesequences of SEQ ID NO 7, 9, 17, 25, 27 or 29 with conservative aminoacid substitutions.

In certain embodiments the polynucleotide or nucleic acid is DNA. Inother embodiments, a polynucleotide of the present invention is RNA, forexample, in the form of messenger RNA (mRNA). RNA of the presentinvention may be single stranded or double stranded.

Recombinant Methods

Fusion proteins of the invention may be obtained, for example, bysolid-state peptide synthesis (e.g. Merrifield solid phase synthesis) orrecombinant production. For recombinant production one or morepolynucleotide encoding the fusion protein (fragment), e.g., asdescribed above, is isolated and inserted into one or more vectors forfurther cloning and/or expression in a host cell. Such polynucleotidemay be readily isolated and sequenced using conventional procedures. Inone embodiment a vector, preferably an expression vector, comprising oneor more of the polynucleotides of the invention is provided. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing the coding sequence of a fusionprotein (fragment) along with appropriate transcriptional/translationalcontrol signals. These methods include in vitro recombinant DNAtechniques, synthetic techniques and in vivo recombination/geneticrecombination. See, for example, the techniques described in Maniatis etal., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring HarborLaboratory, N.Y. (1989); and Ausubel et al., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience,N.Y (1989). The expression vector can be part of a plasmid, virus, ormay be a nucleic acid fragment. The expression vector includes anexpression cassette into which the polynucleotide encoding the fusionprotein (fragment) (i.e. the coding region) is cloned in operableassociation with a promoter and/or other transcription or translationcontrol elements. As used herein, a “coding region” is a portion ofnucleic acid which consists of codons translated into amino acids.Although a “stop codon” (TAG, TGA, or TAA) is not translated into anamino acid, it may be considered to be part of a coding region, ifpresent, but any flanking sequences, for example promoters, ribosomebinding sites, transcriptional terminators, introns, 5′ and 3′untranslated regions, and the like, are not part of a coding region. Twoor more coding regions can be present in a single polynucleotideconstruct, e.g. on a single vector, or in separate polynucleotideconstructs, e.g. on separate (different) vectors. Furthermore, anyvector may contain a single coding region, or may comprise two or morecoding regions, e.g. a vector of the present invention may encode one ormore polypeptides, which are post- or co-translationally separated intothe final proteins via proteolytic cleavage. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or unfused to a polynucleotide encoding thefusion protein (fragment) of the invention, or variant or derivativethereof. Heterologous coding regions include without limitationspecialized elements or motifs, such as a secretory signal peptide or aheterologous functional domain. An operable association is when a codingregion for a gene product, e.g. a polypeptide, is associated with one ormore regulatory sequences in such a way as to place expression of thegene product under the influence or control of the regulatorysequence(s). Two DNA fragments (such as a polypeptide coding region anda promoter associated therewith) are “operably associated” if inductionof promoter function results in the transcription of mRNA encoding thedesired gene product and if the nature of the linkage between the twoDNA fragments does not interfere with the ability of the expressionregulatory sequences to direct the expression of the gene product orinterfere with the ability of the DNA template to be transcribed. Thus,a promoter region would be operably associated with a nucleic acidencoding a polypeptide if the promoter was capable of effectingtranscription of that nucleic acid. The promoter may be a cell-specificpromoter that directs substantial transcription of the DNA only inpredetermined cells. Other transcription control elements, besides apromoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operably associated with thepolynucleotide to direct cell-specific transcription. Suitable promotersand other transcription control regions are disclosed herein. A varietyof transcription control regions are known to those skilled in the art.These include, without limitation, transcription control regions, whichfunction in vertebrate cells, such as, but not limited to, promoter andenhancer segments from cytomegaloviruses (e.g. the immediate earlypromoter, in conjunction with intron-A), simian virus 40 (e.g. the earlypromoter), and retroviruses (such as, e.g. Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit â-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas inducible promoters (e.g. promoters inducible tetracyclins).Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from viral systems (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence). Theexpression cassette may also include other features such as an origin ofreplication, and/or chromosome integration elements such as retrovirallong terminal repeats (LTRs), or adeno-associated viral (AAV) invertedterminal repeats (ITRs).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. For example, if secretionof the fusion is desired, DNA encoding a signal sequence may be placedupstream of the nucleic acid encoding a fusion protein of the inventionor a fragment thereof. According to the signal hypothesis, proteinssecreted by mammalian cells have a signal peptide or secretory leadersequence which is cleaved from the mature protein once export of thegrowing protein chain across the rough endoplasmic reticulum has beeninitiated. Those of ordinary skill in the art are aware thatpolypeptides secreted by vertebrate cells generally have a signalpeptide fused to the N-terminus of the polypeptide, which is cleavedfrom the translated polypeptide to produce a secreted or “mature” formof the polypeptide. In certain embodiments, the native signal peptide,e.g. an immunoglobulin heavy chain or light chain signal peptide isused, or a functional derivative of that sequence that retains theability to direct the secretion of the polypeptide that is operablyassociated with it. Alternatively, a heterologous mammalian signalpeptide, or a functional derivative thereof, may be used. For example,the wild-type leader sequence may be substituted with the leadersequence of human tissue plasminogen activator (TPA) or mouseβ-glucuronidase. The amino acid and nucleotide sequences of an exemplarysecretory signal peptide are shown in SEQ ID NOs 35 and 36,respectively.

DNA encoding a short protein sequence that could be used to facilitatelater purification (e.g. a histidine tag) or assist in labeling thefusion protein may be included within or at the ends of the fusionprotein (fragment) encoding polynucleotide.

In a further embodiment, a host cell comprising one or morepolynucleotides of the invention is provided. In certain embodiments ahost cell comprising one or more vectors of the invention is provided.The polynucleotides and vectors may incorporate any of the features,singly or in combination, described herein in relation topolynucleotides and vectors, respectively. In one such embodiment a hostcell comprises (e.g. has been transformed or transfected with) a vectorcomprising a polynucleotide that encodes (part of) a fusion protein ofthe invention. As used herein, the term “host cell” refers to any kindof cellular system which can be engineered to generate the fusionproteins of the invention or fragments thereof. Host cells suitable forreplicating and for supporting expression of fusion proteins are wellknown in the art. Such cells may be transfected or transduced asappropriate with the particular expression vector and large quantitiesof vector containing cells can be grown for seeding large scalefermenters to obtain sufficient quantities of the fusion protein forclinical applications. Suitable host cells include prokaryoticmicroorganisms, such as E. coli, or various eukaryotic cells, such asChinese hamster ovary cells (CHO), insect cells, or the like. Forexample, polypeptides may be produced in bacteria in particular whenglycosylation is not needed. After expression, the polypeptide may beisolated from the bacterial cell paste in a soluble fraction and can befurther purified. In addition to prokaryotes, eukaryotic microbes suchas filamentous fungi or yeast are suitable cloning or expression hostsfor polypeptide-encoding vectors, including fungi and yeast strainswhose glycosylation pathways have been “humanized”, resulting in theproduction of a polypeptide with a partially or fully humanglycosylation pattern. See Gerngross, Nat Biotech 22, 1409-1414 (2004),and Li et al., Nat Biotech 24, 210-215 (2006). Suitable host cells forthe expression of (glycosylated) polypeptides are also derived frommulticellular organisms (invertebrates and vertebrates). Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains have been identified which may be used in conjunction withinsect cells, particularly for transfection of Spodoptera frugiperdacells. Plant cell cultures can also be utilized as hosts. See e.g. U.S.Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants). Vertebrate cells may also be used as hosts. Forexample, mammalian cell lines that are adapted to grow in suspension maybe useful. Other examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line(293 or 293T cells as described, e.g., in Graham et al., J Gen Virol 36,59 (1977)), baby hamster kidney cells (BHK), mouse sertoli cells (TM4cells as described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)),monkey kidney cells (CV1), African green monkey kidney cells (VERO-76),human cervical carcinoma cells (HELA), canine kidney cells (MDCK),buffalo rat liver cells (BRL 3A), human lung cells (W138), human livercells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (asdescribed, e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68(1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host celllines include Chinese hamster ovary (CHO) cells, including dhfr⁻ CHOcells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); andmyeloma cell lines such as YO, NS0, P3X63 and Sp2/0. For a review ofcertain mammalian host cell lines suitable for protein production, see,e.g., Yazaki and, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo,ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003). Host cells includecultured cells, e.g., mammalian cultured cells, yeast cells, insectcells, bacterial cells and plant cells, to name only a few, but alsocells comprised within a transgenic animal, transgenic plant or culturedplant or animal tissue. In one embodiment, the host cell is a eukaryoticcell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO)cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., Y0,NS0, Sp20 cell).

Standard technologies are known in the art to express foreign genes inthese systems. Cells expressing a polypeptide comprising either theheavy or the light chain of an antibody, may be engineered so as to alsoexpress the other of the antibody chains such that the expressed productis an antibody that has both a heavy and a light chain.

In one embodiment, a method of producing a fusion protein according tothe invention is provided, wherein the method comprises culturing a hostcell comprising a polynucleotide encoding the fusion protein, asprovided herein, under conditions suitable for expression of the fusionprotein, and recovering the fusion protein from the host cell (or hostcell culture medium).

In the fusion proteins of the invention, the components (IgG-classantibody and IL-10 molecule) are genetically fused to each other. Fusionproteins can be designed such that its components are fused directly toeach other or indirectly through a linker sequence. The composition andlength of the linker may be determined in accordance with methods wellknown in the art and may be tested for efficacy. Additional sequencesmay also be included to incorporate a cleavage site to separate theindividual components of the fusion protein if desired, for example anendopeptidase recognition sequence.

In certain embodiments the fusion proteins of the invention comprise atleast an antibody variable region capable of binding to an antigen suchas FAP. Variable regions can form part of and be derived from naturallyor non-naturally occurring antibodies and fragments thereof. Methods toproduce polyclonal antibodies and monoclonal antibodies are well knownin the art (see e.g. Harlow and Lane, “Antibodies, a laboratory manual”,Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodiescan be constructed using solid phase-peptide synthesis, can be producedrecombinantly (e.g. as described in U.S. Pat. No. 4,186,567) or can beobtained, for example, by screening combinatorial libraries comprisingvariable heavy chains and variable light chains (see e.g. U.S. Pat. No.5,969,108 to McCafferty).

Any animal species of antibody can be used in the invention.Non-limiting antibodies useful in the present invention can be ofmurine, primate, or human origin. If the antibody is intended for humanuse, a chimeric form of antibody may be used wherein the constantregions of the antibody are from a human. A humanized or fully humanform of the antibody can also be prepared in accordance with methodswell known in the art (see e.g. U.S. Pat. No. 5,565,332 to Winter).Humanization may be achieved by various methods including, but notlimited to (a) grafting the non-human (e.g., donor antibody) CDRs ontohuman (e.g. recipient antibody) framework and constant regions with orwithout retention of critical framework residues (e.g. those that areimportant for retaining good antigen binding affinity or antibodyfunctions), (b) grafting only the non-human specificity-determiningregions (SDRs or a-CDRs; the residues critical for the antibody-antigeninteraction) onto human framework and constant regions, or (c)transplanting the entire non-human variable domains, but “cloaking” themwith a human-like section by replacement of surface residues. Humanizedantibodies and methods of making them are reviewed, e.g., in Almagro andFransson, Front Biosci 13, 1619-1633 (2008), and are further described,e.g., in Riechmann et al., Nature 332, 323-329 (1988); Queen et al.,Proc Natl Acad Sci USA 86, 10029-10033 (1989); U.S. Pat. Nos. 5,821,337,7,527,791, 6,982,321, and 7,087,409; Jones et al., Nature 321, 522-525(1986); Morrison et al., Proc Natl Acad Sci 81, 6851-6855 (1984);Morrison and Oi, Adv Immunol 44, 65-92 (1988); Verhoeyen et al., Science239, 1534-1536 (1988); Padlan, Molec Immun 31(3), 169-217 (1994);Kashmiri et al., Methods 36, 25-34 (2005) (describing SDR (a-CDR)grafting); Padlan, Mol Immunol 28, 489-498 (1991) (describing“resurfacing”); Dall'Acqua et al., Methods 36, 43-60 (2005) (describing“FR shuffling”); and Osbourn et al., Methods 36, 61-68 (2005) and Klimkaet al., Br J Cancer 83, 252-260 (2000) (describing the “guidedselection” approach to FR shuffling). Particular antibodies according tothe invention are human antibodies. Human antibodies and human variableregions can be produced using various techniques known in the art. Humanantibodies are described generally in van Dijk and van de Winkel, CurrOpin Pharmacol 5, 368-74 (2001) and Lonberg, Curr Opin Immunol 20,450-459 (2008). Human variable regions can form part of and be derivedfrom human monoclonal antibodies made by the hybridoma method (see e.g.Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)). Human antibodies and humanvariable regions may also be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge (see e.g. Lonberg, Nat Biotech 23, 1117-1125(2005). Human antibodies and human variable regions may also begenerated by isolating Fv clone variable region sequences selected fromhuman-derived phage display libraries (see e.g., Hoogenboom et al. inMethods in Molecular Biology 178, 1-37 (O'Brien et al., ed., HumanPress, Totowa, N.J., 2001); and McCafferty et al., Nature 348, 552-554;Clackson et al., Nature 352, 624-628 (1991)). Phage typically displayantibody fragments, either as single-chain Fv (scFv) fragments or as Fabfragments. A detailed description of the preparation of antibodies byphage display can be found in the Examples appended to WO 2012/020006,which is incorporated herein by reference in its entirety.

In certain embodiments, the antibodies comprised in the fusion proteinsof the present invention are engineered to have enhanced bindingaffinity according to, for example, the methods disclosed in PCTpublication WO 2012/020006 (see Examples relating to affinitymaturation) or U.S. Pat. Appl. Publ. No. 2004/0132066, the entirecontents of which are hereby incorporated by reference. The ability ofthe antibody of the invention to bind to a specific antigenicdeterminant can be measured either through an enzyme-linkedimmunosorbent assay (ELISA) or other techniques familiar to one of skillin the art, e.g. surface plasmon resonance technique (Liljeblad, et al.,Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley,Endocr Res 28, 217-229 (2002)). Competition assays may be used toidentify an antibody that competes with a reference antibody for bindingto a particular antigen, e.g. an antibody that competes with the 4G8antibody for binding to FAP. In certain embodiments, such a competingantibody binds to the same epitope (e.g. a linear or a conformationalepitope) that is bound by the reference antibody. Detailed exemplarymethods for mapping an epitope to which an antibody binds are providedin Morris (1996) “Epitope Mapping Protocols”, in Methods in MolecularBiology vol. 66 (Humana Press, Totowa, N.J.). In an exemplarycompetition assay, immobilized antigen (e.g. FAP) is incubated in asolution comprising a first labeled antibody that binds to the antigen(e.g. 4G8 antibody) and a second unlabeled antibody that is being testedfor its ability to compete with the first antibody for binding to theantigen. The second antibody may be present in a hybridoma supernatant.As a control, immobilized antigen is incubated in a solution comprisingthe first labeled antibody but not the second unlabeled antibody. Afterincubation under conditions permissive for binding of the first antibodyto the antigen, excess unbound antibody is removed, and the amount oflabel associated with immobilized antigen is measured. If the amount oflabel associated with immobilized antigen is substantially reduced inthe test sample relative to the control sample, then that indicates thatthe second antibody is competing with the first antibody for binding tothe antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manualch.14 (Cold Spring. Harbor Laboratory, Cold Spring Harbor, N.Y.).

Fusion proteins prepared as described herein may be purified byart-known techniques such as high performance liquid chromatography, ionexchange chromatography, gel electrophoresis, affinity chromatography,size exclusion chromatography, and the like. The actual conditions usedto purify a particular protein will depend, in part, on factors such asnet charge, hydrophobicity, hydrophilicity etc., and will be apparent tothose having skill in the art. For affinity chromatography purificationan antibody, ligand, receptor or antigen can be used to which the fusionprotein binds. For example, for affinity chromatography purification offusion proteins of the invention, a matrix with protein A or protein Gmay be used. Sequential Protein A or G affinity chromatography and sizeexclusion chromatography can be used to isolate a fusion proteinessentially as described in the Examples. The purity of the fusionprotein can be determined by any of a variety of well known analyticalmethods including gel electrophoresis, high pressure liquidchromatography, and the like. For example, the fusion proteins expressedas described in the Examples were shown to be intact and properlyassembled as demonstrated by reducing and non-reducing SDS-PAGE (seee.g. FIG. 2, 5).

Compositions, Formulations, and Routes of Administration

In a further aspect, the invention provides pharmaceutical compositionscomprising any of the fusion proteins provided herein, e.g., for use inany of the below therapeutic methods. In one embodiment, apharmaceutical composition comprises any of the fusion proteins providedherein and a pharmaceutically acceptable carrier. In another embodiment,a pharmaceutical composition comprises any of the fusion proteinsprovided herein and at least one additional therapeutic agent, e.g. asdescribed below.

Further provided is a method of producing a fusion protein of theinvention in a form suitable for administration in vivo, the methodcomprising (a) obtaining a fusion protein according to the invention,and (b) formulating the fusion protein with at least onepharmaceutically acceptable carrier, whereby a preparation of fusionprotein is formulated for administration in vivo.

Pharmaceutical compositions of the present invention comprise atherapeutically effective amount of one or more fusion protein dissolvedor dispersed in a pharmaceutically acceptable carrier. The phrases“pharmaceutical or pharmacologically acceptable” refers to molecularentities and compositions that are generally non-toxic to recipients atthe dosages and concentrations employed, i.e. do not produce an adverse,allergic or other untoward reaction when administered to an animal, suchas, for example, a human, as appropriate. The preparation of apharmaceutical composition that contains at least one fusion protein andoptionally an additional active ingredient will be known to those ofskill in the art in light of the present disclosure, as exemplified byRemington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference. Moreover, for animal (e.g.,human) administration, it will be understood that preparations shouldmeet sterility, pyrogenicity, general safety and purity standards asrequired by FDA Office of Biological Standards or correspondingauthorities in other countries. Preferred compositions are lyophilizedformulations or aqueous solutions. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, buffers, dispersionmedia, coatings, surfactants, antioxidants, preservatives (e.g.antibacterial agents, antifungal agents), isotonic agents, absorptiondelaying agents, salts, preservatives, antioxidants, proteins, drugs,drug stabilizers, polymers, gels, binders, excipients, disintegrationagents, lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the therapeutic orpharmaceutical compositions is contemplated.

The composition may comprise different types of carriers depending onwhether it is to be administered in solid, liquid or aerosol form, andwhether it need to be sterile for such routes of administration asinjection. Fusion proteins of the present invention (and any additionaltherapeutic agent) can be administered intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostatically, intrasplenically, intrarenally,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, intratumorally, intramuscularly,intraperitoneally, subcutaneously, subconjunctivally, intravesicularly,mucosally, intrapericardially, intraumbilically, intraocularly, orally,topically, locally, by inhalation (e.g. aerosol inhalation), injection,infusion, continuous infusion, localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in cremes, in lipid compositions(e.g. liposomes), or by other method or any combination of the forgoingas would be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference). Parenteral administration, inparticular intravenous injection, is most commonly used foradministering polypeptide molecules such as the fusion proteins of theinvention.

Parenteral compositions include those designed for administration byinjection, e.g. subcutaneous, intradermal, intralesional, intravenous,intraarterial intramuscular, intrathecal or intraperitoneal injection.For injection, the fusion proteins of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks' solution, Ringer's solution, or physiological saline buffer.The solution may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the fusion proteinsmay be in powder form for constitution with a suitable vehicle, e.g.,sterile pyrogen-free water, before use. Sterile injectable solutions areprepared by incorporating the fusion proteins of the invention in therequired amount in the appropriate solvent with various of the otheringredients enumerated below, as required. Sterility may be readilyaccomplished, e.g., by filtration through sterile filtration membranes.Generally, dispersions are prepared by incorporating the varioussterilized active ingredients into a sterile vehicle which contains thebasic dispersion medium and/or the other ingredients. In the case ofsterile powders for the preparation of sterile injectable solutions,suspensions or emulsion, the preferred methods of preparation arevacuum-drying or freeze-drying techniques which yield a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered liquid medium thereof. The liquid mediumshould be suitably buffered if necessary and the liquid diluent firstrendered isotonic prior to injection with sufficient saline or glucose.The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein. Suitable pharmaceuticallyacceptable carriers include, but are not limited to: buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Aqueous injectionsuspensions may contain compounds which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, dextran,or the like. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl cleats or triglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(18th Ed. Mack Printing Company, 1990). Sustained-release preparationsmay be prepared. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe polypeptide, which matrices are in the form of shaped articles, e.g.films, or microcapsules. In particular embodiments, prolonged absorptionof an injectable composition can be brought about by the use in thecompositions of agents delaying absorption, such as, for example,aluminum monostearate, gelatin or combinations thereof.

In addition to the compositions described previously, the fusionproteins may also be formulated as a depot preparation. Such long actingformulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the fusion proteins may be formulated with suitablepolymeric or hydrophobic materials (for example as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

Pharmaceutical compositions comprising the fusion proteins of theinvention may be manufactured by means of conventional mixing,dissolving, emulsifying, encapsulating, entrapping or lyophilizingprocesses. Pharmaceutical compositions may be formulated in conventionalmanner using one or more physiologically acceptable carriers, diluents,excipients or auxiliaries which facilitate processing of the proteinsinto preparations that can be used pharmaceutically. Proper formulationis dependent upon the route of administration chosen.

The fusion proteins may be formulated into a composition in a free acidor base, neutral or salt form. Pharmaceutically acceptable salts aresalts that substantially retain the biological activity of the free acidor base. These include the acid addition salts, e.g. those formed withthe free amino groups of a proteinaceous composition, or which areformed with inorganic acids such as for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric ormandelic acid. Salts formed with the free carboxyl groups can also bederived from inorganic bases such as for example, sodium, potassium,ammonium, calcium or ferric hydroxides; or such organic bases asisopropylamine, trimethylamine, histidine or procaine. Pharmaceuticalsalts tend to be more soluble in aqueous and other protic solvents thanare the corresponding free base forms.

Therapeutic Methods and Compositions

Any of the fusion proteins provided herein may be used in therapeuticmethods. For use in therapeutic methods, fusion proteins of theinvention would be formulated, dosed, and administered in a fashionconsistent with good medical practice. Factors for consideration in thiscontext include the particular disorder being treated, the particularmammal being treated, the clinical condition of the individual patient,the cause of the disorder, the site of delivery of the agent, the methodof administration, the scheduling of administration, and other factorsknown to medical practitioners.

In one aspect, fusion proteins of the invention for use as a medicamentare provided. In further aspects, fusion proteins of the invention foruse in treating a disease are provided. In certain embodiments, fusionproteins of the invention for use in a method of treatment are provided.In one embodiment, the invention provides a fusion protein as describedherein for use in the treatment of a disease in an individual in needthereof. In certain embodiments, the invention provides a fusion proteinfor use in a method of treating an individual having a diseasecomprising administering to the individual a therapeutically effectiveamount of the fusion protein. In certain embodiments the disease to betreated is an inflammatory disease. Exemplary inflammatory diseasesinclude inflammatory bowel disease (e.g. Crohn's disease or ulcerativecolitis) and rheumatoid arthritis. In a particular embodiment thedisease is inflammatory bowel disease or rheumatoid arthritis,particularly inflammatory bowel disease, more particularly Crohn'sdisease or ulcerative colitis. In certain embodiments the method furthercomprises administering to the individual a therapeutically effectiveamount of at least one additional therapeutic agent, e.g., ananti-inflammatory agent if the disease to be treated is an inflammatorydisease. An “individual” according to any of the above embodiments is amammal, preferably a human.

In a further aspect, the invention provides for the use of a fusionprotein of the invention in the manufacture or preparation of amedicament for the treatment of a disease in an individual in needthereof. In one embodiment, the medicament is for use in a method oftreating a disease comprising administering to an individual having thedisease a therapeutically effective amount of the medicament. In certainembodiments the disease to be treated is an inflammatory disease. In aparticular embodiment the disease is inflammatory bowel disease orrheumatoid arthritis, particularly inflammatory bowel disease, moreparticularly Crohn's disease or ulcerative colitis. In one embodiment,the method further comprises administering to the individual atherapeutically effective amount of at least one additional therapeuticagent, e.g., an anti-inflammatory agent if the disease to be treated isan inflammatory disease. An “individual” according to any of the aboveembodiments may be a mammal, preferably a human.

In a further aspect, the invention provides a method for treating adisease in an individual, comprising administering to said individual atherapeutically effective amount of a fusion protein of the invention.In one embodiment a composition is administered to said individual,comprising a fusion protein of the invention in a pharmaceuticallyacceptable form. In certain embodiments the disease to be treated is aninflammatory disease. In a particular embodiment the disease isinflammatory bowel disease or rheumatoid arthritis, particularlyinflammatory bowel disease, more particularly Crohn's disease orulcerative colitis. In certain embodiments the method further comprisesadministering to the individual a therapeutically effective amount of atleast one additional therapeutic agent, e.g. an anti-inflammatory agentif the disease to be treated is an inflammatory disease. An “individual”according to any of the above embodiments may be a mammal, preferably ahuman.

The fusion proteins of the invention are also useful as diagnosticreagents. The binding of a fusion proteins to an antigenic determinantcan be readily detected e.g. by a label attached to the fusion proteinor by using a labeled secondary antibody specific for the fusion proteinof the invention.

In some embodiments, an effective amount of a fusion protein of theinvention is administered to a cell. In other embodiments, atherapeutically effective amount of a fusion protein of the invention isadministered to an individual for the treatment of disease.

For the prevention or treatment of disease, the appropriate dosage of afusion protein of the invention (when used alone or in combination withone or more other additional therapeutic agents) will depend on the typeof disease to be treated, the route of administration, the body weightof the patient, the type of fusion protein, the severity and course ofthe disease, whether the fusion protein is administered for preventiveor therapeutic purposes, previous or concurrent therapeuticinterventions, the patient's clinical history and response to the fusionprotein, and the discretion of the attending physician. The practitionerresponsible for administration will, in any event, determine theconcentration of active ingredient(s) in a composition and appropriatedose(s) for the individual subject. Various dosing schedules includingbut not limited to single or multiple administrations over varioustime-points, bolus administration, and pulse infusion are contemplatedherein.

The fusion protein is suitably administered to the patient at one timeor over a series of treatments. Depending on the type and severity ofthe disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) offusion protein can be an initial candidate dosage for administration tothe patient, whether, for example, by one or more separateadministrations, or by continuous infusion. One typical daily dosagemight range from about 1 μg/kg to 100 mg/kg or more, depending on thefactors mentioned above. For repeated administrations over several daysor longer, depending on the condition, the treatment would generally besustained until a desired suppression of disease symptoms occurs. Oneexemplary dosage of the fusion protein would be in the range from about0.005 mg/kg to about 10 mg/kg. In other non-limiting examples, a dosemay also comprise from about 1 μg/kg body weight, about 5 μg/kg bodyweight, about 10 μg/kg body weight, about 50 μg/kg body weight, about100 μg/kg body weight, about 200 μg/kg body weight, about 350 μg/kg bodyweight, about 500 μg/kg body weight, about 1 mg/kg body weight, about 5mg/kg body weight, about 10 mg/kg body weight, about 50 mg/kg bodyweight, about 100 mg/kg body weight, about 200 mg/kg body weight, about350 mg/kg body weight, about 500 mg/kg body weight, to about 1000 mg/kgbody weight or more per administration, and any range derivable therein.In non-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg body weight to about 100 mg/kg bodyweight, about 5 μg/kg body weight to about 500 mg/kg body weight etc.,can be administered, based on the numbers described above. Thus, one ormore doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10 mg/kg (or anycombination thereof) may be administered to the patient. Such doses maybe administered intermittently, e.g. every week or every three weeks(e.g. such that the patient receives from about two to about twenty, ore.g. about six doses of the fusion protein). An initial higher loadingdose, followed by one or more lower doses may be administered. However,other dosage regimens may be useful. The progress of this therapy iseasily monitored by conventional techniques and assays.

The fusion proteins of the invention will generally be used in an amounteffective to achieve the intended purpose. For use to treat or prevent adisease condition, the fusion proteins of the invention, orpharmaceutical compositions thereof, are administered or applied in atherapeutically effective amount. Determination of a therapeuticallyeffective amount is well within the capabilities of those skilled in theart, especially in light of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays, such as cell culture assays. Adose can then be formulated in animal models to achieve a circulatingconcentration range that includes the IC₅₀ as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the fusion proteins which are sufficient to maintaintherapeutic effect. Usual patient dosages for administration byinjection range from about 0.1 to 50 mg/kg/day, typically from about 0.5to 1 mg/kg/day. Therapeutically effective plasma levels may be achievedby administering multiple doses each day. Levels in plasma may bemeasured, for example, by HPLC.

In cases of local administration or selective uptake, the effectivelocal concentration of the fusion protein may not be related to plasmaconcentration. One having skill in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.

A therapeutically effective dose of the fusion proteins described hereinwill generally provide therapeutic benefit without causing substantialtoxicity. Toxicity and therapeutic efficacy of a fusion protein can bedetermined by standard pharmaceutical procedures in cell culture orexperimental animals. Cell culture assays and animal studies can be usedto determine the LD₅₀ (the dose lethal to 50% of a population) and theED₅₀ (the dose therapeutically effective in 50% of a population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, which can be expressed as the ratio LD₅₀/ED₅₀. Fusion proteinsthat exhibit large therapeutic indices are preferred. In one embodiment,the fusion protein according to the present invention exhibits a hightherapeutic index. The data obtained from cell culture assays and animalstudies can be used in formulating a range of dosages suitable for usein humans. The dosage lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon a variety of factors,e.g., the dosage form employed, the route of administration utilized,the condition of the subject, and the like. The exact formulation, routeof administration and dosage can be chosen by the individual physicianin view of the patient's condition (see, e.g., Fingl et al., 1975, in:The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, incorporatedherein by reference in its entirety).

The attending physician for patients treated with fusion proteins of theinvention would know how and when to terminate, interrupt, or adjustadministration due to toxicity, organ dysfunction, and the like.Conversely, the attending physician would also know to adjust treatmentto higher levels if the clinical response were not adequate (precludingtoxicity). The magnitude of an administered dose in the management ofthe disorder of interest will vary with the severity of the condition tobe treated, with the route of administration, and the like. The severityof the condition may, for example, be evaluated, in part, by standardprognostic evaluation methods. Further, the dose and perhaps dosefrequency will also vary according to the age, body weight, and responseof the individual patient.

Other Agents and Treatments

The fusion proteins of the invention may be administered in combinationwith one or more other agents in therapy. For instance, a fusion proteinof the invention may be co-administered with at least one additionaltherapeutic agent. The term “therapeutic agent” encompasses any agentadministered to treat a symptom or disease in an individual in need ofsuch treatment. Such additional therapeutic agent may comprise anyactive ingredients suitable for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. In certain embodiments, an additional therapeuticagent is an anti-inflammatory agent.

Such other agents are suitably present in combination in amounts thatare effective for the purpose intended. The effective amount of suchother agents depends on the amount of fusion protein used, the type ofdisorder or treatment, and other factors discussed above. The fusionproteins are generally used in the same dosages and with administrationroutes as described herein, or about from 1 to 99% of the dosagesdescribed herein, or in any dosage and by any route that isempirically/clinically determined to be appropriate.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate compositions), and separate administration, in which case,administration of the fusion protein of the invention can occur priorto, simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant.Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a fusion protein of the invention. The label or packageinsert indicates that the composition is used for treating the conditionof choice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an fusion protein of the invention; and (b) a second containerwith a composition contained therein, wherein the composition comprisesa further therapeutic agent. The article of manufacture in thisembodiment of the invention may further comprise a package insertindicating that the compositions can be used to treat a particularcondition. Alternatively, or additionally, the article of manufacturemay further comprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook etal., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions. DNA sequences were determined by double strand sequencing.General information regarding the nucleotide sequences of humanimmunoglobulins light and heavy chains is given in: Kabat, E. A. et al.,(1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIHPublication No 91-3242.

Gene Synthesis

Desired gene segments were either generated by PCR using appropriatetemplates or synthesized at Geneart AG (Regensburg, Germany) fromsynthetic oligonucleotides and PCR products by automated gene synthesis.The gene segments flanked by singular restriction endonuclease cleavagesites were cloned into standard cloning/sequencing vectors. The plasmidDNA was purified from transformed bacteria and the concentrationdetermined by UV spectroscopy. The DNA sequences of the subcloned genefragments were confirmed by DNA sequencing. Gene segments were designedwith suitable restriction sites to allow subcloning into the respectiveexpression vectors. All constructs were designed with a 5′-end DNAsequence coding for a leader peptide (MGWSCIILFLVATATGVHS) which targetsproteins for secretion in eukaryotic cells.

Cloning of Antibody-IL-10 Fusion Constructs

The amplified DNA fragments of heavy and light chain V-domains wereinserted in frame either into the human IgG₁ or the Fab constant heavychain or the human constant light chain containing respective recipientmammalian expression vector. Heavy chains and light chains were alwaysencoded on separate plasmids. Whereas the plasmids coding for the lightchains are identical for IgG-based and Fab-based IL-10 fusionconstructs, the plasmids encoding the heavy chains for the Fab-basedconstructs contain, depending on the format, one or two VH—CH1 domainsalongside with the respective IL-10 portion. In the case where the Fabheavy chain plasmid comprises two VH—CH1 domains (tandem Fab intermittedby a single chain IL-10 dimer or by an engineered monomeric IL-10(Josephson et al., J Biol Chem 275, 13552-7 (2000)), the two V-domainshad to be inserted in a two-step cloning procedure using differentcombinations of restriction sites for each of them. The IL-10 portionsof these constructs were always cloned in frame with the heavy chains ofthese antibodies using a (G₄S)₃ 15-mer linker between the C-terminus ofthe Fab or IgG heavy chain and the N-terminus of the cytokine,respectively. Only the IgG-IL-10 format (FIG. 1A) comprises a (G₄S)₄20-mer linker between the C-terminus of the IgG heavy chain and theN-terminus of the cytokine. The C-terminal lysine residue of the IgGheavy chain was removed upon addition of the connector. For the singlechain IL-10, a (G₄S)₄ 20-mer linker was inserted between the two IL-10chains. In the case of two different IgG heavy chains with only one ofthem fused to IL-10, two heavy chain plasmids needed to be constructedand transfected for heterodimerization facilitated by a knob-into-holemodification in the IgG CH3 domains. The “hole” heavy chain connected tothe IL-10 portion carried the Y349C, T366S, L368A and Y407V mutations inthe CH3 domain, whereas the unfused “knob” heavy chain carried the S354Cand T366W mutations in the CH3 domain (EU numbering). To abolish FcγRbinding/effector function and prevent FcR co-activation, the followingmutations were introduced into the CH2 domain of each of the IgG heavychains: L234A, L235A and P329G (EU numbering). The expression of theantibody-IL-10 fusion constructs was driven by an MPSV promoter andtranscription was terminated by a synthetic polyA signal sequencelocated downstream of the CDS. In addition to the expression cassette,each vector contained an EBV oriP sequence for autonomous replication inEBV-EBNA expressing cell lines.

Preparation of Antibody-IL-10 Fusion Proteins

Details about the generation, affinity maturation and characterizationof antigen binding moieties directed to FAP can be found in the Examples(particularly Example 2-6 (preparation) and 7-13 (characterization))appended to PCT publication no. WO 2012/020006, which is incorporatedherein by reference in its entirety. As described therein, variousantigen binding domains directed to FAP have been generated by phagedisplay, including the ones designated 4G8 and 4B9 used in the followingexamples.

Antibody IL-10 fusion constructs as used in the examples were producedby co-transfecting exponentially growing HEK293-EBNA cells with themammalian expression vectors using a calcium phosphate-transfection.Alternatively, HEK293 EBNA cells growing in suspension were transfectedby polyethylenimine (PEI) with the expression vectors. All FAP-targetingantibody-IL-10 fusion constructs based on clones 4G8 and 4B9 can bepurified by affinity chromatography using a protein A matrix.

Briefly, FAP-targeted constructs fused to IL-10, single chain (sc) IL-10or IL-10M1 were purified by a method composed of one affinitychromatography step (protein A) followed by size exclusionchromatography (Superdex 200, GE Healthcare). The protein A column wasequilibrated in 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5,supernatant was loaded, and the column was washed with 20 mM sodiumphosphate, 20 mM sodium citrate (optionally with or without 500 mMsodium chloride), pH 7.5, followed by a wash with 13.3 mM sodiumphosphate, 20 mM sodium citrate, 500 mM sodium chloride, pH 5.45 in caseFBS was present in the supernatant. A third wash with 10 mM MES, 50 mMsodium chloride pH 5 was optionally performed. The fusion constructswere eluted with 20 mM sodium citrate, 100 mM sodium chloride, 100 mMglycine, pH 3. The eluted fractions were pooled and polished by sizeexclusion chromatography in the final formulation buffer which waseither 25 mM potassium phosphate, 125 mM sodium chloride, 100 mM glycinepH 6.7 or 20 mM histidine, 140 mM NaCl pH6.0.

The protein concentration of purified antibody-IL-10 fusion constructswas determined by measuring the optical density (OD) at 280 nm, usingthe molar extinction coefficient calculated on the basis of the aminoacid sequence. Purity, integrity and monomeric state of the fusionconstructs were analyzed by SDS-PAGE in the presence and absence of areducing agent (5 mM 1,4-dithiotreitol) and stained with Coomassie blue(SimpleBlue™ SafeStain, Invitrogen). The NuPAGE® Pre-Cast gel system(Invitrogen) was used according to the manufacturer's instructions(4-20% Tris-glycine gels or 3-12% Bis-Tris). Alternatively, reduced andnon-reduced antibody-IL-10 fusion constructs were analyzed using aLabChip GX (Caliper) according to manufacturer's specifications. Theaggregate content of immunoconjugate samples was analyzed using aSuperdex 200 10/300GL analytical size-exclusion column (GE Healthcare)with 2 mM MOPS, 150 mM NaCl, 0.02% NaN₃, pH 7.3 running buffer, or aTSKgel G3000 SW XL column in 25 mM K2HPO4, 125 mM NaCl, 200 mM arginine,0.02% NaN3, pH 6.7 running buffer at 25° C.

Results of the purifications and subsequent analysis for the differentconstructs are shown in FIG. 2-8. The IgG-IL-10 construct exhibitedseveral production advantages over the other IL-10 fusion formats.Firstly, in comparison to the Fab-IL-10 format, the IL-10 homodimer isanchored within the same antibody molecule. Consequently, uponproduction, no monomeric IL-10 molecules can occur as seen for theFab-IL-10 format for which after affinity chromatography, monomeric anddimeric protein species were observed with only the dimer being thedesired active product (compare FIG. 2B and FIG. 6B). Secondly, incontrast to heterodimeric IgG-based formats comprising a knob-into-holemodification (e.g. IgG-scIL-10 and IgG-IL-10M1), the IgG-IL-10 constructcomprises two identical heavy chains. This avoids undesired byproductslike hole-hole or knob-knob homodimers.Affinity-Determination by SPR

Kinetic rate constants (k_(on) and k_(off)) as well as affinity (K_(D))of antibody-IL-10 fusion constructs to FAP from three different species(human, murine and cynomolgus) and to human IL-10R1 were measured bysurface plasmon resonance (SPR) using a ProteOn XPR36 (BioRad)instrument with PBST running buffer (10 mM phosphate, 150 mM sodiumchloride pH 7.4, 0.005% Tween 20) at 25° C. To determine the affinitiesto FAP, the target protein was captured via its H6-tag by a covalentlyimmobilized anti-H6 antibody (FIG. 9A). Briefly, anti-penta His IgG(Qiagen #34660, mouse monoclonal antibody) was immobilized at highlevels (up to ˜5.000 RU) at 30 μl/min onto separate vertical channels ofa GLM chip by simultaneously activating all channels for 5 min with afreshly prepared mixture of1-ethyl-3-(3-dimethylaminopropyl)-carboiimide (EDC) andN-hydroxysuccinimide (sNHS), subsequently injecting 15 μg/ml anti-pentaHis IgG in 10 mM sodium acetate buffer pH 4.5 for 180 sec. Channels wereblocked using a 5-min injection of ethanolamine. H6-tagged FAP fromdifferent species (see SEQ ID NOs 81, 83 and 85) was captured from a 5μg/ml dilution in running buffer along the vertical channels for 60 s at30 μl/min to achieve ligand densities between ˜250 and 600 RU. In aone-shot kinetic assay set-up (OSK), antibody-IL-10 fusion constructswere injected as analytes along the horizontal channels in a five-folddilution series ranging from 50 to 0.08 nM at 100 μl/min. Associationphase was recorded for 180 s, dissociation phase for 600 s. In case ofinteractions exhibiting very slow off-rates, recording of off-rates wasextended up to 1800 s in order to observe the dissociation of thecomplex. However, in some instances, fitting of these off-rates wasstill not possible so an estimate of 1×10⁻⁵ 1/s was used for calculationof K_(D). Running buffer (PBST) was injected along the sixth channel toprovide an “in-line” blank for referencing. Association rates (k_(on))and dissociation rates (k_(off)) were calculated using a simple 1:1Langmuir binding model (ProteOn Manager software version 2.1) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (K_(D)) was calculated as the ratiok_(off)/k_(on). Regeneration was performed by two pulses of 10 mMglycine pH 1.5 and 50 mM NaOH for 30 s at 100 μl/min in the horizontalorientation to dissociate the anti-penta His IgG from captured FAP andbound antibody-IL-10 fusion constructs.

To measure the interaction between the antibody-IL-10 fusion constructsand the human IL-10R1, an NLC chip was used for immobilization of thebiotinylated receptor (FIG. 9B). Between 400 and 1600 RU of humanIL-10R1 (see SEQ ID NO: 87) were captured on the neutravidin-derivatizedchip matrix along vertical channels at a concentration of 10 μg/ml and aflow rate of 30 μl/sec for varying contact times. Binding tobiotinylated human IL10R1 was measured at six different analyteconcentrations (50, 10, 2, 0.4, 0.08, 0 nM) by injections in horizontalorientation at 100 μl/min, recording the association rate for 180 s, thedissociation rate for 600 s. Running buffer (PBST) was injected alongthe sixth channel to provide an “in-line” blank for referencing.Association rates (k_(on)) and dissociation rates (k_(off)) werecalculated using a simple 1:1 Langmuir binding model (ProteOn Managersoftware version 2.1) by simultaneously fitting the association anddissociation sensorgrams. The equilibrium dissociation constant (K_(D))was calculated as the ratio k_(off)/k_(on). As human IL-10R1 could notbe regenerated without a loss of activity, the two subsequent steps ofligand capture and analyte injection were performed channel per channelusing a freshly immobilized sensorchip surface for every interaction.

Table 1 and 2 show a summary of kinetic rate and equilibrium constantsfor antibody-IL-10 fusion constructs based on anti-FAP clone 4G8 or 4B9,respectively, binding to FAP from different species and to humanIL-10R1.

TABLE 1 Summary of kinetic rate and equilibrium constants for antibodyfusions based on anti-FAP clone 4G8. Binding to FAP from differentspecies and to human IL-10R1. hu IL-10R1 hu FAP mu FAP cyno FAP 4G8(k_(on), k_(off), K_(D)) (k_(on), k_(off), K_(D)) (k_(on), k_(off),K_(D)) (k_(on), k_(off), K_(D)) IgG-IL-10 9.96 × 10⁵ 1/Ms 3.04 × 10⁶1/Ms 1.55 × 10⁶ 1/Ms 3.66 × 10⁶ 1/Ms 2.46 × 10⁻⁵ 1/s 1.24 × 10⁻⁴ 1/s1.00 × 10⁻⁵ 1/s est. 1.06 × 10⁻⁴ 1/s 2.47 × 10⁻¹¹ M 4.07 × 10⁻¹¹ M 6.45× 10⁻¹² M 2.90 × 10⁻¹¹ M IgG-scIL-10 n.d. because of n.d. because ofn.d. because of n.d. because of heterogeneity heterogeneityheterogeneity heterogeneity of protein of protein of protein of proteinIgG-IL- 3.64 × 10⁵ 1/Ms 2.26 × 10⁶ 1/Ms 1.99 × 10⁶ 1/Ms 3.75 × 10⁶ 1/Ms10M1 2.96 × 10⁻⁴ 1/s 7.93 × 10⁻⁵ 1/s 1.00 × 10⁻⁵ 1/s est. 1.28 × 10⁻⁴1/s 8.15 × 10⁻¹⁰ M 3.52 × 10⁻¹¹ M 5.03 × 10⁻¹² M 3.41 × 10⁻¹¹ M IgG-(IL-1.58E+06 1/Ms 3.09 × 10⁶ 1/Ms 1.70 × 10⁶ 1/Ms 3.45 × 10⁶ 1/Ms 10M1)₂3.79 × 10⁻⁵ 1/s 7.76 × 10⁻⁵ 1/s 1.12 × 10⁻⁵ 1/s 1.80 × 10⁻⁴ 1/s 2.40 ×10⁻¹¹ M 2.51 × 10⁻¹¹ M 6.57 × 10⁻¹² M 5.21 × 10⁻¹¹ M Fab-IL-10 1.32 ×10⁶ 1/Ms 3.24 × 10⁶ 1/Ms 1.77 × 10⁶ 1/Ms 3.55 × 10⁶ 1/Ms 8.23 × 10⁻⁵ 1/s1.69 × 10⁻⁴ 1/s 1.00 × 10⁻⁵ 1/s est. 1.29 × 10⁻⁴ 1/s 6.24 × 10⁻¹¹ M 5.21× 10⁻¹¹ M 5.65 × 10⁻¹² M 3.64 × 10⁻¹¹ M Fab-scIL- 1.30 × 10⁶ 1/Ms 4.01 ×10⁶ 1/Ms 1.80 × 10⁶ 1/Ms 4.03 × 10⁶ 1/Ms 10-Fab 9.55 × 10⁻⁵ 1/s 2.18 ×10⁻⁴ 1/s 1.00 × 10⁻⁵ 1/s est. 2.19 × 10⁻⁴ 1/s 7.33 × 10⁻¹¹ M 5.43 ×10⁻¹¹ M 5.56 × 10⁻¹² M 5.44 × 10⁻¹¹ M Fab-IL-  3.7 × 10⁵ 1/Ms 3.66 × 10⁶1/Ms 1.52 × 10⁶ 1/Ms 3.84 × 10⁶ 1/Ms 10M1-Fab  4.2 × 10⁻⁴ 1/s 2.04 ×10⁻⁴ 1/s 1.00 × 10⁻⁵ 1/s est. 2.42 × 10⁻⁴ 1/s  1.1 × 10⁻⁹ M 5.57 × 10⁻¹¹M 5.58 × 10⁻¹² M 6.29 × 10⁻¹¹ M

TABLE 2 Summary of kinetic rate and equilibrium constants for antibodyfusions based on anti-FAP clone 4B9. Binding to FAP from differentspecies and to human IL-10R1. hu IL-10R1 hu FAP mu FAP cyno FAP 4B9(k_(on), k_(off), K_(D)) (k_(on), k_(off), K_(D)) (k_(on), k_(off),K_(D)) (k_(on), k_(off), K_(D)) IgG-IL-10 8.24 × 10⁵ 1/Ms 3.81 × 10⁶1/Ms 2.12 × 10⁶ 1/Ms 5.47 × 10⁶ 1/Ms 3.91 × 10⁻⁵ 1/s 4.03 × 10⁻⁵ 1/s1.24 × 10⁻⁴ 1/s 2.86 × 10⁻⁵ 1/s 4.75 × 10⁻¹¹ M 1.06 × 10⁻¹¹ M 5.83 ×10⁻¹¹ M 5.22 × 10⁻¹² M IgG-(IL- 1.80 × 10⁶ 1/Ms 5.80 × 10⁶ 1/Ms 2.97 ×10⁶ 1/Ms 6.40 × 10⁶ 1/Ms 10M1)₂ 3.39 × 10⁻⁵ 1/s 9.73 × 10⁻⁵ 1/s 1.09 ×10⁻⁴ 1/s 7.77 × 10⁻⁵ 1/s 1.88 × 10⁻¹¹ M 1.68 × 10⁻¹¹ M 3.69 × 10⁻¹¹ M1.21 × 10⁻¹¹ M Fab-IL-10 2.15 × 10⁶ 1/Ms 5.47 × 10⁶ 1/Ms 2.68 × 10⁶ 1/Ms4.16 × 10⁶ 1/Ms 4.57 × 10⁻⁵ 1/s 5.72 × 10⁻⁶ 1/s 6.27 × 10⁻⁵ 1/s 7.27 ×10⁻⁵ 1/s 2.12 × 10⁻¹¹ M 1.05 × 10⁻¹² M 2.34 × 10⁻¹¹ M 1.75 × 10⁻¹¹ MFab-scIL- 1.73 × 10⁶ 1/Ms 4.74 × 10⁶ 1/Ms 2.45 × 10⁶ 1/Ms 4.93 × 10⁶1/Ms 10-Fab 9.58 × 10⁻⁵ 1/s 3.11 × 10⁻⁵ 1/s 7.40 × 10⁻⁵ 1/s 3.35 × 10⁻⁵1/s 5.53 × 10⁻¹¹ M 6.56 × 10⁻¹² M 3.03 × 10⁻¹¹ M 6.79 × 10⁻¹² M

Wild type (wt) IL-10 cytokine, not fused to an antibody but C-terminallyH6-tagged, in our hands showed a K_(D) of 52 pM for human IL-10R1(k_(on), 2.5×10⁶ 1/Ms, k_(off) 1.3×10⁻⁴ 1/s). For the antibody-IL-10fusion constructs based on the dimeric cytokine, the avidities toIL-10R1 were comparable to the unfused cytokine and also two-digit pM(ranging from 18 to 73 pM). This showed that this cytokine toleratesN-terminal fusions to antibodies or fragments thereof without asignificant loss of avidity for human IL-10R1. In contrast, theantibody-IL-10 fusion constructs based on the monomeric cytokine did notshow the avidity effect of the dimeric IL-10 fusions and thus theiraffinities to the receptor were in the three-digit pM or one-digit nMrange (815 pM and 1.1 nM, respectively). Binding to FAP depends on therespective antibody, with clone 4B9 showing higher affinity/avidity tohuman and cynomolgus FAP, whereas clone 4G8 exhibits higheraffinity/avidity to murine FAP. In fact, the avidity of the 4G8 antibodyto murine FAP was so strong that it was impossible to determine thedissociation rate of the complex under the applied conditions. Theinteraction between IL-10 and IL-10R1 is of high affinity (avidity)ranging from ˜35-200 pM (Moore, K. W. et al., Annu. Rev. Immunol. 19,683-765 (2001)). For the constructs comprising a dimeric IL-10 portionor two independent monomers, the fusion to the antibody does not seem toalter the affinity significantly (˜19-73 pM). However, for the monomericIL-10 fusion constructs, this strength of binding was dramaticallyreduced, most likely, because there is no avidity effect as occurs forthe dimeric cytokine or two monomers fused to the same IgG. Ideally, theaffinity of the antibody-IL-10 fusion constructs to the target FAPshould be higher than that for the high affinity cytokine receptorIL-10R1 in order to achieve efficient targeting to tissues where FAP isexpressed. Despite the high affinity between IL-10 and IL-10R1, theaffinities to the target FAP exhibited by the molecules based on theIgG-IL-10 format are still higher: clone 4B9 IgG-IL-10 (48 pM to IL-10R1vs. 11 pM to human FAP) and clone 4G8 (25 pM to IL-10R1 vs. 6 pM tomurine FAP), respectively. These affinities to IL-10R1 as well as to FAPseem to be suitable for achieving efficient targeting toFAP-overexpressing tissues and IL-10R1 does not seem to represent a sinkfor these molecules.

Suppression of LPS-Induced Production of Pro-Inflammatory Cytokines byPrimary Monocytes

For functional characterization and differentiation between IgG or Fabbased FAP-targeted IL-10 constructs the potency of these molecules wasassessed in different in vitro assays. For example the efficacy tosuppress LPS-induced production of pro-inflammatory cytokines by primarymonocytes was measured. For this experiment, 200 ml of heparinizedperipheral blood (obtained from healthy volunteers, Medical Servicesdepartment, Roche Diagnostics GmbH, Penzberg, Germany) was separated byFicoll Hypaque density gradient followed by negative isolation ofmonocytes (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany,#130-091-153). Purified monocytes were seeded in 96-well F cell cultureplates (Costar/Corning Life Sciences, Amsterdam, The Netherlands; #3596)at 5×10⁴ cells/well in medium (RPMI 1640 [Gibco/Invitrogen, Darmstadt,Germany, cat. no. #10509-24] supplemented with 10% human serum, 2 mML-glutamine [Gibco, #25030], and Pen/Strep).

All antibody-IL-10 fusion proteins were tested (a) in solution and (b)in an experimental setting, in which recombinant human FAP (c_(fin)=1μg/ml) was coated overnight at 4° C. onto the plate (alternatively,60-90 min at room temperature) and the antibody-IL-10 fusion proteinsimmobilized by binding to the coated FAP.

For set-up (a), cells were stimulated directly after seeding with 100ng/ml LPS (Sigma-Aldrich/Nunc, Taufkirchen, Germany, # L3129) in thepresence or absence of titrated amounts (normally, 0-500 nM) of theindicated antibody-fusion constructs or recombinant wild type humanIL-10 as positive control. For set-up (b) unbound FAP was removed aftercoating, and plates were blocked with medium (see above) for 1 h at roomtemperature, before incubation with IL-10 constructs for an additionalhour. Thereafter, plates were washed with medium, before monocytes wereadded into the culture together with an appropriate stimulus (100 ng/mlLPS).

For all experiments, cells were incubated for 24 h at 37° C. and 5% CO₂.Supernatants were collected (optionally stored at −20/−80° C.) andtested for cytokine production using CBA Flex Sets for IL-1β, IL-6,G-CSF, and/or TNFα (BD Biosciences, Heidelberg, Germany, #558279,#558276, #558326 and #558299). Plates were measured with a FACS Arrayand analyzed using FCAP software (both purchased from BD).

As shown in FIG. 10, the in vitro potency of 4G8 Fab-IL-10 and IgG-IL-10in the suppression of pro-inflammatory cytokines IL-1β, IL-6, and TNFαwas comparable in set-up (a) (FIG. 10 D, E, F). In contrast, in set-up(b) the IgG-based format demonstrated superior potency compared toFab-IL-10 (FIG. 10 A, B, C). The corresponding EC50 values [nM] areshown in Table 3. The EC50 values of the IgG-IL-10 construct in set-up(b) were similar to the ones of recombinant wt human IL-10 (which couldonly be tested in set-up (a)).

TABLE 3 EC50 values of 4G8-IgG-IL-10 and 4G8-Fab-IL-10 for suppressionof cytokine production by monocytes (donor 1). EC50 [nM] set-up (a) EC50[nM] set-up (b) (solution) (immobilized) sample hIL-6 hIL-1β hTNFα hIL-6hIL-1β hTNFα hu wt IL-10 0.010 0.009 0.002 not applicable/testedIgG-IL-10 0.054 0.049 0.017 0.002 0.001 0.001 Fab-IL-10 0.083 0.0590.023 0.103 0.085 0.017

This result was reproduced in an independent experiment, using twodifferent blood donors (FIGS. 11A-11F and 11G-11L, Table 4 and 5). Inthis experiment again the IgG-based targeted IL-10 construct wassignificantly superior to the Fab-based molecule in the suppression ofall three cytokines tested, as indicated by the EC50 values obtained inset-up (b). In set-up (a), all molecules were comparable.

TABLE 4 EC50 values of 4G8-IgG-IL-10 and 4G8-Fab-IL-10 for suppressionof cytokine production by monocytes (donor 2). EC50 [nM] set-up (a) EC50[nM] set-up (b) (solution) (immobilized) sample hIL-6 hIL-1β hTNFα hIL-6hIL-1β hTNFα hu wt IL-10 0.006 0.002 0.002 not applicable/testedIgG-IL-10 0.039 0.015 0.011 0.001 0.0002 0.0002 Fab-IL-10 0.061 0.0300.024 0.060 0.023 0.017

TABLE 5 EC50 values of 4G8-IgG-IL-10 and 4G8-Fab-IL-10 for suppressionof cytokine production by monocytes (donor 3). EC50 [nM] set-up (a) EC50[nM] set-up (b) (solution) (immobilized) Sample hIL-6 hIL-1β hTNFα hIL-6hIL-1β hTNFα hu wt IL-10 0.004 0.003 0.001 not applicable/testedIgG-IL-10 0.036 0.020 0.019 0.001 0.0002 0.0002 Fab-IL-10 0.065 0.0520.052 0.057 0.036 0.025

In a further experiment, the potency of Fab and IgG based IL-10constructs in the suppression of IL-6 production by monocytes was againassessed, and compared to wt IL-10 as well as untargeted Fab-IL-10 andIgG-IL-10 constructs, which do not bind to FAP (FIG. 12 and Table 6).Again, 4G8-IgG-IL-10 was found to be more efficient in the suppressionof IL-6 production in the experimental set-up (b) compared to4G8-Fab-IL-10, while untargeted constructs caused suppression only atthe highest concentrations. In contrast, in set-up (a), potency of allconstructs was comparable.

TABLE 6 EC50 values of 4G8-IgG-IL-10 and 4G8-Fab-IL-10 for suppressionof IL-6 production by monocytes (donor 4). EC50 [nM] IL-6 set-up (a)set-up (b) Sample (solution) (immobilized) hu wt IL-10 0.007 not testedIgG-IL-10 0.123 0.002 Fab-IL-10 0.078 0.081 germline IgG-IL-10 0.166 notcalculable germline Fab-IL-10 0.152 not calculable

As the concentration of recombinant human FAP used for coating in theprevious assays might reflect an artificial or non-physiologiccondition, the amount of coated FAP was titrated (c_(fin) between 015and 5 μg/ml) and its impact on EC50 values assessed in the experimentalset-up (b).

As shown in FIG. 13, overall there is no drastic difference in theratios of EC50 values for IgG- and Fab-based constructs. At allconcentrations, the IgG-IL-10 construct was more potent in theinhibition of IL-6 induction (FIG. 13 and Table 7 and 8). Theconcentration of coated FAP did, however, influence the outcome of theexperiments as with decreasing concentrations the EC50 values generallyincreased, which might reflect the amount of constructs immobilized onthe microtiter plate (Table 8; for the Fab-based construct a cytokinereduction was observed at the lowest FAP concentrations, but an EC50could not be calculated). Interestingly, at high FAP concentrations (5μg/ml) an increase in the total amount of secreted IL-6 was detected(FIG. 14).

TABLE 7 EC50 values of 4G8-IgG-IL-10 and human wild-type IL-10 (insolution) for suppression of IL-6 production by monocytes (donor 5).Sample hIL-6 EC50 [nM] 4G8-IgG-IL-10 0.066 hu wt IL-10 0.010

TABLE 8 EC50 values of 4G8-IgG-IL-10 and 4G8-Fab-IL-10, immobilized ondifferent concentrations of coated FAP, for suppression of IL-6production by monocytes (donor 5). hIL-6 EC50 [nM] FAP conc.4G8-IgG-IL-10 4G8-Fab-IL-10 0.25 μg/ml 0.019 — 0.5 μg/ml 0.001 — 1 μg/ml0.002 0.029 5 μg/ml 0.0004 0.016

In a further experiment, IL-10 fusion constructs comprising a differentFAP targeting domain, affinity-matured anti-FAP antibody variant 4B9,was tested. Again, the in vitro potency of the constructs in suppressionof LPS-induced IL-6 production by monocytes was assessed in experimentalset-up (a) and (b).

FIG. 15 shows that for 4B9-based constructs the IgG-IL-10 molecules weresuperior to the Fab-IL-10 constructs in suppression of IL-6 productionin experimental set-up (a) (and comparable in set-up (b)). CorrespondingEC50 values are shown in Table 9. In general, 4B9 and 4G8 constructsdemonstrated similar potency.

TABLE 9 EC50 values of 4G8 and 4B9-based IgG-IL-10 and Fab-IL-10 forsuppression of IL-6 production by monocytes (donor 7). EC50 [nM] IL-6Sample Set-up (a) Set-up (b) hu wt IL-10 0.008 not tested 4G8 IgG-IL-10not tested 0.009 4G8 Fab-IL-10 not tested 0.065 4B9 IgG-IL-10 0.0380.002 4B9 Fab-IL-10 0.063 not calculable

In a further series of experiments, 4G8-based IgG-IL-10, Fab-IL-10,Fab-IL-10M1-Fab and IgG-IL-10M1 constructs were compared. Suppression ofLPS-induced production of pro-inflammatory cytokines IL-6, IL-1β andTNFα by monocytes was assessed in experimental set-up (a) and (b). Theresults of these experiments are shown in Tables 10-12 (three differentdonors). As in previous experiments, IgG-IL-10 was the most potentconstruct, particularly in experimental set-up (b).

TABLE 10 EC50 values of 4G8 IgG-IL-10, 4G8 Fab-IL-10, 4G8Fab-IL-10M1-Fab and 4G8 IgG-IL- 10M1 fusion proteins for suppression ofcytokine production by monocytes (donor 1). EC50 [nM] set-up (a) EC50[nM] set-up (b) (solution) (immobilized) Sample hIL-6 hIL-1β hTNFα hIL-6hIL-1β hTNFα hu wt IL-10 0.010 0.009 0.002 not tested not tested nottested IgG-IL-10 0.054 0.049 0.017 0.002 0.001 0.001 Fab-IL-10 0.0860.059 0.023 0.103 0.085 0.017 Fab-IL-10M1- not not not not not not Fabcalculable calculable calculable calculable calculable calculableIgG-IL-10M1 not not not not not not calculable calculable calculablecalculable calculable calculable

TABLE 11 EC50 values of 4G8 IgG-IL-10, 4G8 Fab-IL-10, 4G8Fab-IL-10M1-Fab and 4G8 IgG-IL- 10M1 fusion proteins for suppression ofcytokine production by monocytes (donor 2). EC50 [nM] set-up (a) EC50[nM] set-up (b) (solution) (immobilized) Sample hIL-6 hIL-1β hTNFα hIL-6hIL-1β hTNFα hu wt IL-10 0.006 0.002 0.002 not tested not tested nottested IgG-IL-10 0.039 0.015 0.011 0.001 0.0002 0.0002 Fab-IL-10 0.0610.030 0.024 0.060 0.023 0.017 Fab-IL-10M1- not not not not 3.339 2.847Fab calculable calculable calculable calculable IgG-IL-10M1 not not not0.723 0.140 0.059 calculable calculable calculable

TABLE 12 EC50 values of 4G8 IgG-IL-10, 4G8 Fab-1L-10, 4G8Fab-IL-10M1-Fab and 4G8 IgG-IL-10M1 fusion proteins for suppression ofcytokine production by monocytes (donor 3). EC50 [nM] set-up (a) EC50[nM] set-up (b) (solution) (immobilized) Sample hIL-6 hIL-1β hTNFα hIL-6hIL-1β hTNFα hu wt IL-10 0.004 0.003 0.001 not tested not tested nottested IgG-IL-10 0.036 0.020 0.019 0.001 0.0002 0.0002 Fab-IL-10 0.0650.052 0.052 0.057 0.036 0.025 Fab-IL-10M1- not not not not 4.713 not Fabcalculable calculable calculable calculable calculable IgG-IL-10M1 not2.890 not 0.254 0.117 0.145 calculable calculable

In still a further series of experiments, 4G8-based Fab-IL-10,Fab-scIL-10-Fab and Fab-IL-10M1-Fab constructs were compared.Suppression of LPS-induced production of IL-6, IL-1β, TNFα and G-CSF bymonocytes was assessed in experimental set-up (a) and (b). The resultsof these experiments are shown in Tables 13-17 (six different donors).The results show, that the construct comprising a dimeric IL-10 moleculeis more potent than the constructs with a scIL-10 or a monomeric IL-10M1molecule.

TABLE 13 EC50 values of 4G8 Fab-IL-10, 4G8 Fab-scIL-10-Fab and 4G8Fab-IL-10M1-Fab fusion proteins for suppression of cytokine productionby monocytes. EC50 [nM] set-up (a) EC50 [nM] set-up (b) (solution)(immobilized) Sample hIL-6 hIL-1β hTNFα hIL-6 hIL-1β hTNFα hu wt IL-100.004 0.004 0.001 not tested not tested not tested Fab-IL-10 0.030 0.0200.007 0.020 0.003 0.001 Fab-scIL-10- 0.110 0.090 0.060 0.200 0.100 0.030Fab Fab-IL-10M1- not not not not not not Fab calculable calculablecalculable calculable calculable calculable

TABLE 14 EC50 values of 4G8 Fab-IL-10, 4G8 Fab-scIL-10-Fab and 4G8Fab-IL-10M1-Fab fusion proteins for suppression of IL-6 production bymonocytes. EC50 [nM] IL-6 supression (solution) Donor Donor Donor DonorDonor Donor Sample #1 #2 #3 #4 #5 #6 Mean Std. dev. hu wt IL-10 0.0040.008 0.004 0.003 0.0003 0.004 0.004 0.002 Fab-IL-10 0.030 n.d. 0.0700.030 0.070 0.260 0.092 0.096 Fab-scIL-10- 0.110 n.d. 0.150 0.110 0.2500.630 0.250 0.220 Fab Fab-IL-10M1- not not not not not not — — Fab calc.calc. calc. calc. calc. calc.

TABLE 15 EC50 values of 4G8 Fab-IL-I0, 4G8 Fab-scIL-10-Fab and 4G8Fab-IL-10M1-Fab fusion proteins for suppression of IL-1β production bymonocytes. EC50 [nM] IL-1β supression (solution) Donor Donor Donor DonorDonor Donor Sample #1 #2 #3 #4 #5 #6 Mean Std. dev. hu wt IL-10 0.0040.006 0.004 0.002 n.d. 0.006 0.004 0.002 Fab-IL-10 0.020 n.d. 0.0500.020 0.050 0.370 0.102 0.150 Fab-scIL-10- 0.090 n.d. 0.110 0.090 0.2701.460 0.404 0.595 Fab Fab-IL-10M1- not not not not not not Fab calc.calc. calc. calc. calc. calc.

TABLE 16 EC50 values of 4G8 Fab-IL-10, 4G8 Fab-scIL-10-Fab and 4G8Fab-IL-10M1-Fab fusion proteins for suppression of G-CSF production bymonocytes. EC50 [nM] G-CSF supression (solution) Donor Donor Donor DonorDonor Donor Sample #1 #2 #3 #4 #5 #6 Mean Std. dev. hu wt IL-10 0.0030.006 0.003 0.003 0.0001 0.003 0.003 0.002 Fab-IL-10 0.010 n.d. 0.0500.010 0.050 260 0.076 0.105 Fab-scIL-10- 0.060 n.d. 0.110 0.060 0.2001.160 0.318 0.474 Fab Fab-IL-10M1- not not not not not not — — Fab calc.calc. calc. calc. calc. calc.

TABLE 17 EC50 values of 4G8 Fab-IL-10, 4G8 Fab-scIL-10-Fab and 4G8Fab-IL-10M1-Fab fusion proteins for suppression of TNFα production bymonocytes. EC50 [nM] TNFα supression (solution) Donor Donor Donor DonorDonor Donor Sample #1 #2 #3 #4 #5 #6 Mean Std. dev. hu wt IL-10 0.0010.002 0.001 0.003 n.d. 0.001 0.002 0.001 Fab-IL-10 0.007 n.d. 0.0400.007 0.040 0.0180 0.055 0.072 Fab-scIL-10- 0.060 n.d. 0.190 0.060 0.0801.660 0.410 0.701 Fab Fab-IL-10M1- not not not not not not — — Fab calc.calc. calc. calc. calc. calc.

Finally, 4B9 and 4G8-based Fab-IL-10 and IgG-(IL-10M1)₂ constructs werecompared. Suppression of LPS-induced production of IL-6 by monocytes wasassessed in experimental set-up (a) and (b). The results of thisexperiment are shown in Table 19. The results show that all constructs,including IgG-(IL-10M1)₂, perform better in set-up (b) than in set-up(a).

TABLE 18 EC50 values of 4B9 IgG-IL-10, 4G8 IgG-IL-10 and 4G8IgG-(IL-10M1)₂ fusion proteins for suppression of IL-6 production bymonocytes. EC50 [nM] set-up (a) EC50 [nM] set-up (b) (solution)(immobilized) Sample hIL-6 hIL-6 hu wt IL-10 0.006 not tested 4B9IgG-IL-10 0.035 0.011 4G8 IgG-IL-10 0.028 0.004 IgG-(IL-10M1)₂ notcalculable 0.039Suppression of IFNγ-Induced Upregulation of MHC-II Molecules on PrimaryMonocytes

For functional characterization and differentiation between IgG and Fabbased FAP-targeted IL-10 constructs their ability to suppressIFNγ-induced MHC-II expression in monocytes was assessed. Similar to thecytokine suppression assay, this experiment was performed with theconstructs either in solution (experimental set-up (a); see above) orimmobilized by binding to FAP coated on the cell culture plate(experimental set-up (b); see above). In principle, monocytes wereisolated and cultured as described above, but stimulated with 250 U/mlIFNγ (BD, #554616) for 24 h. Before stimulation, cells were optionallytreated with recombinant wild-type (wt) IL-10 or the differentantibody-IL-10 fusion constructs. After incubation, cells were detachedby Accutase treatment (PAA, #L11-007) and stained with an anti-HLA-DRantibody (BD, #559866) in PBS containing 3% human serum (Sigma, #4522)to avoid any unspecific FcγR binding before subjecting to final FACSanalysis.

The result of this experiment is shown in Table 19, demonstrating thatfor 4B9-based constructs the IgG-IL-10 molecules were superior to theFab-IL-10 constructs in down-regulation of IFN□-induced MHC-IIexpression on primary monocytes in experimental set-up (b) (andcomparable in set-up (a)).

TABLE 19 EC50 values of 4B9 IgG-IL-10 and 4B9 Fab-IL-10 fordown-regulation of IFN□-induced MHC-II expression on primary monocytes.EC50 [nM] set-up (a) EC50 [nM] set-up (b) sample (solution)(immobilized) Fab-IL-10 0.072 not calculable IgG-IL-10 0.064 0.018 hu wtIL-10 0.004 not testedIL-10 Induced STAT3 Phosphorylation in Isolated Primary Monocytes

As IL-10R signaling leads to phosphorylation of STAT3 several targetedIL-10 constructs and formats were functionally evaluated in a pSTAT3assay using freshly isolated blood monocytes (Finbloom & Winestock, J.Immunol. 1995; Moore et al., Annu. Rev. Immunol. 2001; Mosser & Zhang,Immunological Reviews 2008). Briefly, CD 14⁺ monocytes were untouchedseparated from Ficoll-isolated PBMC of healthy donors as describedabove. Typically, 3−10×10⁵ cells were transferred into FACS tubes in 300μl medium (RPMI1640/10% FCS/L-glutamine/pen/strep) and usually incubatedfor 30 min at 37° C., 5% CO₂, with 0-200/500 nM of wt human IL-10 or theindicated antibody-IL-10 fusion proteins. Then, 300 μl pre-warmed Fixbuffer I (BD Biosciences, #557870) per tube was added, vortexed andincubated for 10 min at 37° C. before cells were washed once with 2 mlPBS/2% FCS and centrifuged at 250×g for 10 min. Subsequently, 300 μlice-cooled Perm Buffer III (BD Biosciences, #558050) per tube was addedfor cell permeabilization and incubated for 30 min on ice before cellswere again washed as described above. Finally, cells were resuspended in100 μl antibody dilution (anti-Stat-3.A647; BD Biosciences, #557815) andincubated for 30 min at 4° C. before cells were washed and processed forFACS analysis.

The EC50 values obtained for the different constructs in this experimentare shown in Tables 20 and 21. The results show that constructscomprising a dimeric IL-10 molecule (Fab- or IgG-based) are more activethan constructs comprising a scIL-10 molecule or a monomeric IL-10M1molecule.

TABLE 20 EC50 values of 4G8-based antibody-IL-10 fusion proteins forIL-10 induced STAT3 phosphorylation in isolated primary monocytes. EC50[nM] pSTAT3 induction Sample Donor 1 Donor 2 Donor 3 hu wt IL-10 0.0290.019 0.021 Fab-IL-10 0.154 0.194 0.087 Fab-scIL-10-Fab 0.557 0.4300.116 Fab-IL-10M1-Fab 8.201 9.012 6.809

TABLE 21 EC50 values of 4B9-based antibody-IL-10 fusion proteins forIL-10 induced STAT3 phosphorylation in isolated primary monocytes.Sample EC50 [nM] pSTAT3 induction hu wt IL-10 0.017 IgG-IL-10 0.130IgG-(IL-10M1)2 0.435Biodistribution of FAP-Targeted and Untargeted Antibody-IL-10 FusionProteins

The tissue biodistribution of FAP-targeted In-111-labeled 4B9 IgG-IL-10,4G8 IgG-IL-10 and untargeted DP47GS IgG-IL 10 was determined at 50 μgper mouse in DBA/1J mice with collagen-induced arthritis reaching apre-determined arthritis score>3 (28 days after the first immunization).Biodistribution was performed at 72 h after i.v. injection ofradiolabeled conjugates in five mice per group.

Results of this experiment are shown in Table 22. Uptake of theuntargeted antibody-IL-10 fusion protein DP47GS IgG-IL-10 in theinflamed joints was significantly lower (p<0.0001) than uptake of thetargeted IgG-IL-10 fusion proteins, indicating that the uptake of 4B9IgG-IL-10 and 4G8 IgG-IL-10 is FAP-mediated. Splenic uptake most likelyis IL-10-mediated, because all three constucts showed similar levels ofsplenic accumulation.

TABLE 22 Uptake of antibody constructs (% injected dose/gram of tissue).Tissue 4B9 IgG-IL-10 4G8 IgG-IL-10 DP47GS IgG-IL-10 inflamed joints 20.7± 1.1 19.6 ± 1.0 8.6 ± 1.0 spleen  6.3 ± 0.4  7.3 ± 0.3 6.7 ± 0.5 blood 4.2 ± 0.5  1.1 ± 0.1 7.3 ± 1.0

To study the effect of the IL-10 on the biodistribution of IgG-IL-10, ina second experiment the biodistribution of In-111-labeled 4G8 IgG-IL-10was compared to that of In-111-labeled 4G8 IgG.

Results of this experiment are shown in Table 23. There was nosignificant difference in accumulation in the inflamed joints between4G8 IgG and 4G8 IgG-IL-10, indicating that IL-10 did not significantlyaffect the targeting of 4G8 IgG to the inflamed sites. Splenic uptake of4G8 IgG1-IL-10 is significantly higher than that of 4G8 IgG (p<0.0001),indicating that uptake in the spleen is partly IL-10 mediated.

TABLE 22 Uptake of antibody constructs (% injected dose/gram of tissue).Tissue 4G8 IgG 4G8 IgG-IL-10 inflamed joints 18.1 ± 1.0 19.6 ± 1.0spleen 2.9 ± 0.2 7.3 ± 0.3 blood 3.9 ± 0.8 1.1 ± 0.1

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

The invention claimed is:
 1. A fusion protein comprising an antibody andan IL-10 molecule, wherein the antibody comprises the heavy chainvariable region of SEQ ID NO: 67 and the light chain variable region ofSEQ ID NO: 69, and wherein the IL-10 molecule comprises SEQ ID NO:1. 2.The fusion protein of claim 1, wherein the fusion protein comprises twoidentical heavy chain polypeptides and two identical light chainpolypeptides.
 3. The fusion protein of claim 2, wherein each of theheavy chain polypeptides comprises an IgG-class antibody heavy chain anda monomeric IL-10 molecule.
 4. The fusion protein of claim 3, whereinthe monomeric IL-10 molecule is fused at its N-terminus to theC-terminus of the IgG-class antibody heavy chain.
 5. The fusion proteinof claim 4 additionally comprising a peptide linker to fuse theN-terminus of the monomeric IL-10 molecule to the C-terminus of theIgG-class antibody heavy chain.
 6. The fusion protein of claim 3,wherein each of the heavy chain polypeptides further comprises a peptidelinker.
 7. The fusion protein of claim 3, wherein the monomeric IL-10molecules comprised in the heavy chain polypeptides form a functionalhomodimeric IL-10 molecule.
 8. The fusion protein of claim 1, whereinthe antibody is an IgG-class antibody and comprises a modification,which modification reduces binding affinity of the antibody to an Fcreceptor, as compared to a corresponding IgG-class antibody without themodification, wherein the IgG-class antibody comprises an amino acidsubstitution at a position according to EU numbering selected from thegroup consisting of 228, 233, 234, 235, 297, 329, and 331 of an antibodyheavy chain.
 9. The fusion protein of claim 8, wherein the Fc receptoris an Fcγ receptor.
 10. The fusion protein of claim 8, wherein the Fcreceptor is an activating Fc receptor.
 11. The fusion protein of claim8, wherein the Fc receptor is selected from the group of FcγRIIIa(CD16a), FcγRI (CD64), FcγRIIa (CD32) and FcαRI (CD89).
 12. The fusionprotein of claim 8, wherein the Fc receptor is FcγIIIa.
 13. The fusionprotein of claim 8, wherein the IgG-class antibody comprises a heavychain comprising an amino acid substitution at position 329 according toEU numbering.
 14. The fusion protein of claim 13, wherein the amino acidsubstitution is P329G.
 15. The fusion protein claim 8, wherein theIgG-class antibody comprises a heavy chain comrising amino acidsubstitutions at positions 234 and 235 according to EU numbering. 16.The fusion protein of claim 15, wherein the amino acid substitutions areL234A and L235A (LALA).
 17. The fusion protein of claim 8, wherein theIgG-class antibody comprises a heavy chain comrising amino acidsubstitutions L234A, L235A and P329G according to EU numbering.
 18. Thefusion protein of claim 1, wherein the antibody is an IgG-classantibody.
 19. The fusion protein of claim 1, wherein the antibody isIgG₁-subclass antibody.
 20. The fusion protein of claim 1, wherein theantibody is a full-length antibody.
 21. The fusion protein of claim 1,wherein the antibody is a human antibody.
 22. A pharmaceuticalcomposition comprising the fusion protein of claim 1 and apharmaceutically acceptable carrier.